Cell extrusion delivery device as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cell delivery, in particular to a cell extrusion delivery device and a preparation method and application thereof. The cell extrusion delivery device provided by the invention comprises a micro-channel and a polymer brush grafted on the surface of the micro-channel, the polymer brush has a lubricating function. By grafting the polymer brush on the surface of the microchannel, the lubricating property of the microchannel can be remarkably improved, the friction force between interfaces in the cell extrusion process is greatly reduced, and then cell damage and channel blockage are reduced; meanwhile, the polymer brush does not obviously influence the aperture change of the micro-channel. By grafting the polymer brush on the surface of the microchannel, cell fragmentation is reduced on the premise of keeping the extrusion delivery efficiency of an original device, and cell activity and expression of related genes cannot be damaged.

Description

Technical Field

[0001] The present invention relates to the technical field of cell delivery, and in particular to a cell squeezing and delivery device and a preparation method and application thereof. Background Art

[0002] The intracellular delivery of exogenous substances is an indispensable technology in biomedical research. It mainly involves the delivery of impermeable cargo molecules (such as nucleic acids, including small interfering RNA or messenger RNA, antibodies, nucleases, and nanomaterials such as quantum dots and nanoantibodies) and drugs into living cells in an efficient and high-throughput manner. This is of vital importance for in-depth monitoring of cell behavior, regulation of cell fate and realization of biological functions. Currently, this technology has been widely used in the fields of cell therapy, cell reprogramming, gene editing and recombinant vaccine production. However, ineffective delivery and cell damage hinder the application of exogenous molecules in cells, especially for key cell types such as immune cells and stem cells. At the same time, the stability of gene expression, cell functionality and cell activity are also key factors restricting the development of research and clinical applications.

[0003] Intracellular delivery technology is developing at an astonishing speed and diversity, aiming to efficiently deliver various exogenous substances while minimizing interference and damage to the physiological state of cells. At present, commonly used technical means include viral vectors, liposomes, and electroporation. Viral vectors have limitations such as high cost, long cycle, harmful inflammation or immune response, and other toxic side effects. In contrast, chemical carriers represented by liposomes have more advantages in safety, but there are still problems such as low delivery efficiency (such as suspended cells), slow delivery, and cytotoxicity. Electroporation is easy to operate, but it requires the use of dedicated electroporation equipment, and under high electric field strength, it may lead to decreased cell viability, damage gene expression, affect cell homeostasis and normal function, and even cause excessive cell death.

[0004] Cell extrusion technology has developed into an important tool for rapid and high-throughput cell loading in biomedical research due to its great potential as an efficient loading method in delivery systems. In microchannels, when the cell diameter exceeds the channel size, they are subjected to the squeezing force exerted by the channel wall on the cells when passing through, inducing local deformation, allowing the cells to compress and adapt to a smaller space. After the cells are subjected to the force, their internal molecular structure and cell membrane will deform. The high deformation of the cells causes the cell volume to change and relax, so that various biomaterials in the surrounding environment can diffuse into various types of cytoplasm. This technology does not rely on exogenous substances and can achieve precise time control and instantaneous delivery, thereby simplifying and accelerating the process of research and clinical application, which is crucial to reducing manufacturing costs. In addition, compared with carrier-mediated delivery, the use of mechanical force to destroy the cell membrane improves the safety of delivery. At present, this technology has promoted the development of cancer vaccines and has entered the stage of multiple clinical trials. Compared with electroporation, extrusion delivery can enhance cell viability, proliferation ability and cytokine secretion, reduce gene dysregulation, and maintain stable cell function.

[0005] Although cell extrusion technology has attracted much attention and has many advantages, it also has a series of challenges, such as easy channel clogging and limited cell loading. This is mainly due to its inherent physical limitations. In particular, when using sticky PDMS microchannels, nonspecific proteins tend to adhere to the PDMS surface once in contact with biomaterials, which has become an important issue restricting the development of PDMS-based microfluidic technology. This clogging phenomenon greatly affects the expected performance of the device, makes delivery results unpredictable, and limits the reuse of channels, which may restrict the clinical application potential of the device. Although high-pressure operation and complex design of multiple parallel channels are the simplest and most feasible methods, these solutions often lead to problems such as reduced cell viability and increased reagent consumption, which in turn pushes up the overall cost. In addition, the existing extrusion technology has many and long pipelines, which increases cell retention, and researchers often find it difficult to implement on a large scale. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a cell squeezing and delivery device and a preparation method and application thereof. The cell squeezing and delivery device provided by the present invention can solve the problems of channel blockage, cell damage and the like in the process of cell squeezing and delivery.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a cell squeezing and delivering device, comprising a microchannel and a polymer brush grafted on the surface of the microchannel; the polymer brush has a lubricating function.

[0009] Preferably, the polymer brush comprises one or more of poly (3-sulfopropyl methacrylate potassium salt), poly (methacryloyloxyethyl trimethyl ammonium chloride) and polyethylene glycol dimethacrylate.

[0010] Preferably, the polymer brush has a single layer or multiple layers.

[0011] Preferably, the microchannel comprises at least one of a microporous membrane, filter paper, a 3D printed microchannel and a microfluidic channel.

[0012] Preferably, the microchannel is made of polydimethylsiloxane or polycarbonate.

[0013] Preferably, the pore size of the microchannel is smaller than the diameter of the cells to be delivered.

[0014] The present invention provides a method for preparing the cell squeezing and delivery device described in the above scheme, comprising the following steps: providing a microchannel;

[0015] Prepare an initiator layer on the surface of the microchannel to obtain a microchannel carrying the initiator layer;

[0016] The microchannel carrying the initiator layer is placed in a solution containing monomers and catalysts to perform atom transfer free radical polymerization to form a polymer brush on the surface of the microchannel, thereby obtaining the cell squeezing and delivery device.

[0017] Preferably, the method for preparing the initiator layer on the surface of the microchannel comprises: preparing the initiator layer on the surface of the microchannel by chemical vapor deposition, or preparing the initiator layer on the surface of the microchannel by vacuum impregnation.

[0018] Preferably, before preparing the initiator layer on the surface of the microchannel, the microchannel is further subjected to a hydroxylation treatment.

[0019] The present invention provides the use of the cell squeezing and delivery device described in the above scheme or the cell squeezing and delivery device prepared by the preparation method described in the above scheme in delivering cells to a destination other than disease diagnosis and treatment.

[0020] The present invention provides a cell extrusion delivery device, comprising a microchannel and a polymer brush grafted on the surface of the microchannel; the polymer brush has a lubricating function. By grafting the polymer brush on the surface of the microchannel, the present invention can significantly improve the lubrication performance of the microchannel, greatly reduce the friction between interfaces during cell extrusion, and thus reduce cell damage, activity reduction and channel blockage; at the same time, the polymer brush will not significantly affect the change in the pore size of the microchannel.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The present invention grafts polymer brushes on the surface of the microchannel, thereby reducing cell fragmentation and not damaging cell activity and the expression of related genes while maintaining the extrusion delivery efficiency of the original device.

[0023] The extrusion delivery device constructed by the present invention significantly reduces channel blockage, reduces cell retention, and enhances device durability.

[0024] The invention has the advantages of simple operation, low cost and easy promotion and transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Flow chart for grafting SPMA polymer brush onto PCTE membrane;

[0026] Figure 2 Schematic diagram of the construction of a PCTE-based delivery device;

[0027] Figure 3 The full XPS spectra of the PCTE film and PCTE-PSPMA coating surface (A), and the change of friction coefficient of the PCTE and PCTE-PSPMA coating surface (B);

[0028] Figure 4 Figure 2. Cell viability was assessed by staining the PCTE membranes and PCTE-PSPMA-treated and untreated cells with trypan blue immediately after treatment.

[0029] Figure 5 The figures are the delivery results of 4kDa FITC-dextran delivered to L929 cells using PCTE membrane and PCTE-PSPMA respectively after 12 hours, and the fluorescence microscopy images of cells without any treatment;

[0030] Figure 6 Dot plots and fluorescence intensity histograms showing the delivery of 4 kDa FITC-dextran to L929 cells by flow cytometry;

[0031] Figure 7 This is a comparison chart of cell retention rates after treatment with PCTE membrane and PCTE-PSPMA;

[0032] Figure 8 SEM images of L929 cells on PCTE membrane and membrane after PCTE-PSPMA treatment;

[0033] Fig. 9 This is the analysis diagram of related gene expression in cells after PCTE membrane and PCTE-PSPMA treatment and without any treatment;

[0034] Fig.10 Figure 2 shows the cytoskeleton analysis after PCTE membrane and PCTE-PSPMA delivery. DETAILED DESCRIPTION

[0035] The invention provides a cell squeezing and delivering device, comprising a microchannel and a polymer brush grafted on the surface of the microchannel; the polymer brush has a lubricating function.

[0036] The present invention does not specifically limit the pore size of the microchannel, and any microchannel known in the art that can be used to deliver cells can be used. In the art, the pore size of the microchannel is smaller than the diameter of the cells to be delivered. In a specific embodiment, for L929 cells, an 8μm pore size can achieve a cell delivery efficiency of more than 90%, but there are many dead cells, so the present invention modifies the pore size of the 8μm microchannel to improve its activity.

[0037] In the present invention, the microchannel may be various microchannels suitable for cell extrusion, such as at least one of a microporous membrane, a filter paper, a 3D printed microchannel and a microfluidic channel.

[0038] In the present invention, the material of the microchannel is preferably polydimethylsiloxane (PDMS) or polycarbonate (PCTE).

[0039] In the present invention, the polymer brush preferably includes one or more of poly (3-sulfopropyl methacrylate potassium salt) (PSPMA), poly (methacryloyloxyethyl trimethyl ammonium chloride) (PMETAC) and polyethylene glycol dimethacrylate (POEGMA). The number of layers of the polymer brush is preferably single layer or multilayer, and in a specific embodiment, it can be 1 layer, 2 layers or 3 layers. When the number of layers of the polymer brush is multilayer, the types of polymer brushes in each layer are preferably different.

[0040] The present invention can significantly improve the lubrication performance of the microchannel by grafting a polymer brush on the surface of the microchannel, greatly reduce the friction between interfaces during cell extrusion, and thus reduce cell damage and channel blockage; at the same time, the polymer brush will not significantly affect the change in the pore size of the microchannel.

[0041] The present invention utilizes polymer brush modification to achieve remarkable effects in improving cell activity, reducing cell fragmentation, reducing cell retention and clogging, and effectively inhibiting nonspecific adsorption of biomolecules.

[0042] The present invention enhances the durability of the device, and provides a reliable approach and a solid foundation for applications such as rapid preparation of engineered cells, gene editing of difficult-to-transfect cells, and cell therapy.

[0043] The present invention provides a method for preparing the cell squeezing and delivery device described in the above scheme, comprising the following steps:

[0044] Providing microchannels;

[0045] Prepare an initiator layer on the surface of the microchannel to obtain a microchannel carrying the initiator layer;

[0046] The microchannel carrying the initiator layer is placed in a solution containing monomers and catalysts to perform atom transfer radical polymerization (ATRP) to form a polymer brush on the surface of the microchannel to obtain the cell squeezing and delivery device.

[0047] In the present invention, unless otherwise specified, the raw materials and equipment used are commercially available products well known in the art.

[0048] The present invention first provides a microchannel.

[0049] The present invention has no special requirements on the source of the microchannel, and commercially available products known in the art can be used. In the embodiment of the present invention, it is specifically provided by a commercially available PCTE membrane.

[0050] After obtaining the microchannel, the present invention prepares an initiator layer on the surface of the microchannel to obtain a microchannel carrying the initiator layer.

[0051] Before preparing the initiator layer on the surface of the microchannel, the present invention preferably first performs a hydroxylation treatment on the microchannel. In the present invention, the hydroxylation treatment preferably includes: treating the microchannel with oxygen plasma for 3 to 5 minutes. The present invention has no special requirements for the conditions of the oxygen plasma treatment, and the treatment conditions well known in the art can be used. The hydroxylation treatment of the present invention makes the surface of the microchannel substrate more hydrophilic, thereby improving the adhesion between the initiator layer and the substrate.

[0052] The present invention has no special requirements for the preparation process of the initiator layer, and a preparation process well known in the art can be used. In a specific embodiment, the initiator layer can be prepared on the surface of the microchannel by chemical vapor deposition (this method obtains an initiator monolayer), or the initiator layer can be prepared on the surface of the microchannel by vacuum impregnation, which will not be exhaustively listed here.

[0053] The present invention does not make special requirements on the specific type of the initiator, and any ATRP initiator known in the art can be used. In the embodiment of the present invention, aminopropyltriethoxysilane (APTES-Br) containing terminal bromine is used as the initiator. In the present invention, the APTES-Br is prepared by a method known in the art, specifically adding 3-aminopropyltriethoxysilane, triethylamine and dichloromethane to a round-bottomed three-necked flask under nitrogen protection, stirring for 10 minutes, placing the flask in an ice-water bath to cool, and then adding 2-bromoisobutyryl bromide dropwise; stirring for 12 hours after the addition is completed, and finally separating and purifying the product to obtain a light yellow oily liquid with an aromatic taste, namely APTES-Br.

[0054] In the present invention, when chemical vapor deposition is used to prepare an initiator monolayer, the present invention has no special requirements for the conditions of the chemical vapor deposition, and the conditions well known in the art can be used to form an initiator monolayer on the surface of the microchannel. In an embodiment of the present invention, the hydroxylated microchannel is placed in a vacuum desiccator containing 20 microliters of initiator, and after vacuuming for 10 minutes, it is allowed to stand for 20 minutes, and the process is repeated twice to form an initiator monolayer on the surface of the microchannel.

[0055] In the present invention, when vacuum impregnation is used to prepare the initiator layer, the present invention preferably prepares the initiator into an initiator solution, immerses the microchannel in the initiator solution, and evacuates the microchannel to allow the initiator to enter the microchannel, and forms an initiator layer on the surface of the microchannel after drying.

[0056] After obtaining the microchannel carrying the initiator layer, the present invention places the microchannel carrying the initiator layer in a solution containing monomers and catalysts to perform atom transfer radical polymerization to form a polymer brush on the surface of the microchannel to obtain the cell extrusion delivery device.

[0057] In the present invention, the monomer is determined according to the polymer brush, and when the polymer brush is polysulfonated polymethacrylate, the monomer is 3-sulfopropyl methacrylate potassium salt (SPMA). The present invention does not specifically limit the type and amount of the catalyst, and a catalyst and amount known in the art can be selected according to the type of the polymer brush.

[0058] In an embodiment of the present invention, when the polymer brush is polysulfonated polymethacrylate, the catalyst is preferably cupric chloride dihydrate; the mass ratio of the monomer to cupric chloride dihydrate is preferably 2:0.02-0.05, more preferably 2:0.035. In the present invention, the solvent of the solution containing the monomer and the catalyst is preferably water, more preferably deionized water. The present invention has no special requirements for the amount of water, as long as the monomer and the catalyst can be completely dissolved. The present invention has no special requirements for the preparation method of the solution containing the monomer and the catalyst, and the monomer and the catalyst can be directly dissolved in water.

[0059] In the present invention, when the polymer brush is poly (3-sulfopropyl methacrylate potassium salt), the solution containing the monomer and the catalyst preferably also includes ascorbic acid and 2,2'-bipyridine. The purpose of adding ascorbic acid in the present invention is to activate the catalyst and promote the ATRP reaction. The present invention does not specifically limit the amount of ascorbic acid used, and the amount well known in the art can be used to activate the catalyst. The present invention adds 2,2'-bipyridine as a ligand to coordinate with the copper catalyst to promote the redox reaction of copper ions between Cu(I) and Cu(II), thereby regulating the generation and elimination of free radicals and controlling the polymerization process. The 2,2'-bipyridine improves the stability and activity of the copper catalyst, optimizes the reaction rate and the molecular weight distribution of the polymer, and ensures the high efficiency and controllability of the polymerization. In the present invention, the mass ratio of the 2,2'-bipyridine to cupric chloride dihydrate is preferably 1:2.

[0060] In the present invention, the atom transfer radical polymerization is preferably carried out under vacuum conditions. In the present invention, the microchannel is preferably placed in a solution containing monomers and catalysts, and then vacuumed for 5 to 10 minutes, and then reacted for 4 to 8 hours.

[0061] After the atom transfer radical polymerization is completed, the present invention rinses the obtained cell extrusion delivery device with deionized water and dries it with nitrogen.

[0062] In the present invention, the polymer brush formed by the above scheme is a single-layer structure. When it is necessary to graft multiple layers of polymer brushes, the present invention preferably places the microchannel grafted with a single-layer polymer brush in a solution containing another monomer and a catalyst for polymerization.

[0063] The present invention provides the use of the cell squeezing and delivery device described in the above scheme or the cell squeezing and delivery device prepared by the preparation method described in the above scheme in delivering cells to a destination other than disease diagnosis and treatment.

[0064] The cell squeezing and delivery device and the preparation method thereof provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0065] Example 1

[0066] according to Figure 1 The process shown is to graft SPMA onto the microchannel surface of the PCTE membrane. The specific preparation process is as follows:

[0067] (1) Preparation of the initiator required for atom transfer radical polymerization (ATRP): aminopropyltriethoxysilane containing terminal bromine (APTES-Br):

[0068] Under nitrogen protection, add 10 mL of 3-aminopropyltriethoxysilane, 7.8 mL of triethylamine and 200 mL of dichloromethane into a round-bottom three-necked flask, stir for 10 minutes, place the flask in an ice-water bath to cool, and then add 5.55 mL of 2-bromoisobutyryl bromide dropwise; stir for 12 hours after the addition is complete. Finally, separate and purify the product to obtain a light yellow oily liquid with an aromatic odor, which is APTES-Br;

[0069] (2) APTES-Br monolayer (mAPTES-Br) was prepared on a PCTE membrane (pore size 8 μm, disk diameter 13 mm, brand: cytiva, model: 10417501) by chemical vapor deposition (CVD):

[0070] The PCTE membrane was treated with oxygen plasma for 3 minutes, and the resulting hydroxylated PCTE membrane was placed in a vacuum desiccator containing 20 μl of APTES-Br, and after evacuation for 10 minutes, it was allowed to stand for 20 minutes for vapor phase chemical deposition. The process was then repeated twice. Finally, a mAPTES-Br layer was obtained on the PCTE membrane.

[0071] (3) Preparation of polymer brushes:

[0072] The PCTE membrane with the initiator deposited was placed horizontally in a 10 ml centrifuge tube, 2 g of SPMA, 5 ml of deionized water, 35 mg of copper chloride dihydrate and 70 mg of 2,2'-bipyridine were placed in a glass bottle and stirred until the monomer and catalyst were completely dissolved, and then 40 mg of ascorbic acid was added and stirred for 2 minutes to activate the catalyst. Then, the resulting mixed solution was poured into the centrifuge tube containing the PCTE membrane, vacuumed for 5 to 10 minutes, and then reacted for 6 hours. Finally, the PCTE membrane was taken out, rinsed with deionized water, and dried with nitrogen to obtain a microchannel delivery device, which was recorded as PCTE-PSPMA.

[0073] In order to confirm whether the polymer brush coating is successfully grafted, the present invention uses XPS technology to determine the surface chemical composition before and after grafting. Figure 3 Figure A shows the full XPS spectrum of the PSPMA polymer brush before and after grafting. In the XPS spectrum, the obvious peak at 168.2 eV corresponds to the sulfonic acid sulfur element, which confirms that the PSPMA brush has been successfully modified on the PCTE surface. The change in surface lubricity is used to verify the change in surface functionality after polymer brush modification. The friction test was carried out with a PDMS ball mill in an aqueous solution at 3N and 2Hz. The results are shown in Figure 2. Figure 3 As shown in Figure B, the PCTE-PSPMA coating surface exhibits a low friction coefficient (about 0.25) and remains relatively stable within 500 friction cycles. These results lay a solid foundation for the successful construction of the PCTE-PSPMA delivery platform.

[0074] Construction and performance evaluation of PCTE-based delivery device

[0075] The specific process is as follows: Figure 2 As shown in FIG. 5 , a PCTE-based high-throughput delivery device was constructed, and the PCTE membrane was installed in a Pop-Top filter holder (Cytiva) and sterilized by high pressure before use.

[0076] The present invention evaluates the effect of the modified polymer brush on the delivery effect. First, the present invention uses the Count-star cell counter to evaluate the immediate viability of L929 cells (NCTC clone 929 [L cell, L-929] mouse fibroblasts) after being treated with a PCTE membrane with a pore size of 8 μm. Specifically, the cells were suspended in a growth medium (composed of MEM (minimum essential medium) + 10% FBS + 1% double antibody (referring to a penicillin-streptomycin mixture)) at a cell concentration of 2×10 5 to 1×10 6 The cell suspension was 1 mL; the syringe was pushed at a speed of 0.5 mL / s to allow the cell suspension to enter the centrifuge tube through PCTE and PCTE-PSPMA; at the same time, a control group (without any treatment) was set up; the above three groups of cell samples were mixed evenly according to the ratio of cell suspension to 0.2% trypan blue solution in a ratio of 1:1, and then 20 μL of the sample was pipetted to The counting plate was inserted into the cell analyzer. Cell viability was evaluated by image analysis of the Count-star automatic cell counter, and the immediate cell viability was calculated as the ratio of the number of trypan blue-rejected cells to the total number of cells in each field of view. The trypan blue staining results showed that compared with PCTE-treated cells (immediate viability of 59%), PCTE-PSPMA-treated cells showed a higher immediate cell viability of 79%, as shown in Figure 2. Figure 4 .

[0077] After trypsin digestion, L929 cells with good growth status were suspended in growth medium (composed of MEM + 10% FBS + 1% double antibody) at a cell concentration of 2×10 5 to 1×10 6 / mL. The cell suspension is 1mL; the 4kDa dextran coupled to fluorescein isothiocyanate (also known as FITC-dextran, the concentration in the cell suspension is 0.5mg / mL) to be delivered is mixed with the cell suspension and transferred to a 5mL syringe; the syringe is pushed at a speed of 0.5mL / s to allow the cell suspension to pass through PCTE and PCTE-PSPMA into the centrifuge tube; at the same time, a control group (cells exposed to the delivery solution but not treated in any way) is set up; after 5 minutes of delivery, PBS is added to wash the cells twice. Centrifuge and resuspend the cells in cell growth medium for culture. The present invention was observed by fluorescence microscopy 12 hours after FITC-dextran delivery, and the results are shown in FIG. Figure 5 Compared with the control group, almost all L929 cells treated with PCTE achieved delivery of fluorescent molecules, but with the production of a large number of dead cells and cell debris. PCTE-PSPMA maintained efficient delivery of fluorescent molecules while maintaining almost no effect on cell activity.

[0078] Flow cytometry evaluation results:

[0079] 12 hours after delivery, the present invention further evaluated the cell activity by flow cytometry. Figure 6 . The results showed that the PCTE-PSPMA-treated cells maintained a high delivery efficiency of 88.11% (specifically calculated with reference to flow cytometry. The present invention sets the top 5% as the threshold of endocytosis in the control group. Then in the cell-treated samples, the sample delivery efficiency is calculated based on the control delivery efficiency). At the same time, cell activity was almost unaffected (the cell activity after PCTE-PSPMA treatment was 90.02%, and the cell activity after PCTE treatment was 59.44%). In addition, the dot plot of flow cytometry showed that the proportion of the main cell population in PCTE-PSPMA-treated cells was significantly increased compared to that in PCTE-treated cells (69.35% for PCTE-PSPMA and 33.47% for PCTE), indicating that PCTE treatment resulted in the production of more cell fragments.

[0080] Evaluation of cell retention results:

[0081] Treatment with PCTE-PSPMA reduced cell retention on the membrane and within the device (see Figure 7 ), which is very important for reducing pore blockage. The cells treated with PCTE-PSPMA had only a 10% cell loss, while the cells treated with PCTE had a 25% cell loss, which will further promote the commercialization of the product. Figure 8 The SEM images of L929 cells on the membranes after PCTE-PSPMA treatment and PCTE membrane treatment show the same results.

[0082] Results of related gene expression analysis in cells:

[0083] Fluorescence quantitative PCR quantifies the expression of genes affecting apoptosis, cytoskeleton and other signals. L929 cells with good growth status were digested with trypsin and suspended in growth medium (MEM + 10% FBS + 1% double antibody). The cell concentration was 2×10 5 to 1×10 6 / mL. The cell suspension was 1mL; the syringe was pushed at a speed of 0.5mL / s to allow the cell suspension to pass through PCTE and PCTE-PSPMA into the centrifuge tube; then it was placed in a 6-well plate and cultured for 36 hours. At the same time, a control group (without any treatment) was set up, samples were collected, and total RNA was extracted. According to the instructions of the TaKaRa reverse transcription DNA removal kit (PrimerScriptTMRT regent Kit with gDNAEraser), the RNA was reverse transcribed to obtain cDNA. After the reverse transcription reaction, TBGreenPremix Ex TaqII (TaKaRa) was selected for real-time fluorescence quantitative PCR, and specific primers were used to detect changes in the mRNA levels of apoptosis-related genes and cytoskeleton genes (CASP3, KRT19 and BCL2). At the same time, the mRNA level of the β-actin gene was set as an internal reference. The results showed that the functionalized PCTE membrane treatment had no significant effect on the expression levels of genes related to cell viability and integrity (ns indicates no significant difference). The results are shown in Fig. 9 .

[0084] Confocal microscopy was used to visualize actin and cell membrane. Cells treated in different ways were fixed in 4% paraformaldehyde for 30 minutes. After washing with PBS, the diluted green fluorescent Alexa Fluor488 wheat germ agglutinin (WGA) conjugate (Invitrogen) was incubated at room temperature for 10 minutes. WGA is a cell-impermeable stain that can selectively bind to N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues, which are usually present on the cell membrane. After labeling, the labeling solution was removed and the cells were washed twice. The samples were permeabilized with 0.1% TritonX-100 for 10 minutes. Alexa Fluor 568-phalloidin (Invitrogen) was diluted with 1% bovine serum albumin (BSA) for fluorescent staining. Phalloidin can bind to F-actin with high selectivity. Incubate in the dark for 60 minutes, wash 3 times with PBS, and image using a laser scanning confocal microscope. The results are shown in Fig.10 It can be seen that the motor proteins in the cells treated with PCTE aggregated around, while the functionalized PCTE membrane treatment also minimized the damage to the cytoskeleton.

[0085] Although the present invention only provides embodiments of PSPMA polymer brushes and PCTE membrane microchannels, based on the same principle, different polymer brushes and multilayer polymer brushes essentially have the same effect in the microchannel, so they are not listed here.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cell squeezing and delivery device, characterized in that: The invention comprises a microchannel and a polymer brush grafted on the surface of the microchannel; the polymer brush has a lubricating function.

2. The cell squeezing and delivery device according to claim 1, characterized in that: The polymer brush includes one or more of poly (3-sulfopropyl) methacrylate potassium salt, poly (methacryloyloxyethyl) trimethyl ammonium chloride and polyethylene glycol dimethacrylate.

3. The cell squeezing and delivery device according to claim 1 or 2, characterized in that: The polymer brush has a single layer or multiple layers.

4. The cell squeezing and delivery device according to claim 1, characterized in that: The microchannel includes at least one of a microporous membrane, filter paper, a 3D printed microchannel and a microfluidic channel.

5. The cell squeezing and delivery device according to claim 1 or 4, characterized in that: The material of the microchannel is polydimethylsiloxane or polycarbonate.

6. The cell squeezing and delivery device according to claim 1 or 4, characterized in that: The pore size of the microchannel is smaller than the diameter of the cells to be delivered.

7. The method for preparing the cell squeezing and delivery device according to any one of claims 1 to 6, characterized in that: The following steps are involved: Providing microchannels; Prepare an initiator layer on the surface of the microchannel to obtain a microchannel carrying the initiator layer; The microchannel carrying the initiator layer is placed in a solution containing monomers and catalysts to perform atom transfer free radical polymerization to form a polymer brush on the surface of the microchannel, thereby obtaining the cell squeezing and delivery device.

8. The preparation method according to claim 7, characterized in that: The method for preparing the initiator layer on the surface of the microchannel includes: preparing the initiator layer on the surface of the microchannel by chemical vapor deposition, or preparing the initiator layer on the surface of the microchannel by vacuum impregnation.

9. The preparation method according to any one of claims 7 to 8, characterized in that: Before preparing the initiator layer on the surface of the microchannel, the method further includes subjecting the microchannel to a hydroxylation treatment.

10. Use of the cell squeezing and delivery device according to any one of claims 1 to 6 or the cell squeezing and delivery device prepared by the preparation method according to any one of claims 7 to 9 in delivering cells to a destination other than disease diagnosis and treatment.

Images