Porous channel device with high toughness and high rigidity for intracellular delivery of therapeutic cells and preparation method of porous channel device

Abstract

The invention relates to a porous channel device with high toughness and high rigidity for therapeutic intracellular delivery of cells and a preparation method of the porous channel device. The preparation method comprises the following steps: mixing a thermoplastic tough high-molecular polymer, a rigid filler and a micron-sized pore-forming agent sacrificial template, heating and curing, removing the template to obtain a porous channel precursor, and packaging by adopting a heat shrink tube and an injector to prepare the porous channel device suitable for intracellular delivery. The porosity of a porous channel is 50%-80%, the aperture range is 5-15 microns, the structure is stable, the toughness is good, the impact strength is larger than 20 kJ / m < 2 >, the high elongation at break is about 100%, the rigidity is high, the elastic modulus is about 50 GPa, and the mechanical strength is high. The device effectively promotes intracellular substance delivery by triggering mechanical stress of cells, has the advantages of high flux and low blockage, and can be suitable for application scenes of various cell types such as gene, protein and drug molecule delivery. The method is simple and convenient in process and low in cost, and has important application value in the fields of high-throughput screening, drug development, gene therapy and the like.

Description

Technical Field

[0001] The present invention relates to intracellular substance delivery technology, and in particular to a porous channel device with high toughness and high rigidity for intracellular delivery of therapeutic cells and a preparation method thereof. The device can induce mechanical stress in cells by physical means, promote the effective entry of macromolecules, drugs or genetic materials into cells, and is widely used in biomedical fields such as cell therapy, gene editing, and drug delivery. Background Art

[0002] With the rapid development of the biomedical field, intracellular delivery technology has become the basis of many core technologies such as cell research, gene therapy, and drug delivery. In gene therapy, the key to successful treatment is to effectively deliver specific genes or therapeutic molecules into target cells. However, existing intracellular delivery technologies generally have some technical bottlenecks, such as cumbersome operation, expensive equipment, and significant cell damage.

[0003] At present, cell delivery methods mainly include viral vector method, electroporation method, liposome method and mechanical shearing method. The viral vector method has efficient gene transfection ability, but there are problems of immune response and high production cost. The electroporation method can temporarily open the cell membrane through an electric field, but it may cause greater damage to the cells, and the operation process is cumbersome. Although the liposome method works well in some cases, it has poor stability and may cause cytotoxicity. The mechanical shearing method is to induce cell membrane deformation by physical means, thereby increasing its permeability to external substances, but the existing shearing device has the disadvantages of low efficiency and complex operation in design. In addition, most of the existing porous channel materials adopt traditional high-cost manufacturing methods, and have poor adaptability to different cell types. Therefore, the present invention also proposes a low-cost and simple preparation method, so that the porous channel device can not only be applied to various cell types, but also simplify the production process, and has good potential for promotion and application.

[0004] In view of the above problems, the present invention proposes a low-cost and simple preparation method, which optimizes the pore size and porosity by combining thermoplastic tough polymer, rigid filler material and micron-level sacrificial template porogen to improve the mechanical stress effect during cell delivery, thereby improving the delivery efficiency and flux and reducing damage to cells. The porous channel device of the present invention can promote the delivery of intracellular substances by simple mechanical extrusion, has high efficiency, economy and easy operation, fills the gap in the prior art, and has wide application potential. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of cell delivery devices in the prior art in terms of delivery efficiency, flux, cell damage control and complexity of the preparation process, and to provide a porous channel device with high toughness and high rigidity for intracellular delivery of therapeutic cells and a preparation method thereof. By optimizing the porous structure and preparation process, the cell delivery efficiency and flux are significantly improved, cell damage is reduced, and it has the advantages of being simple and easy to operate.

[0006] The porous channel device of the present invention promotes the instantaneous increase of cell membrane permeability through mechanical force, thereby achieving efficient delivery of intracellular substances. The porous channel in the device is made of thermoplastic tough polymer (such as polyvinyl butyral (PVB) material), rigid filler (such as nano-SiO 2 ) and a micron-sized porogen sacrificial template (such as water-soluble sodium chloride (NaCl)). To achieve the above purpose, the device used in the present invention includes the following steps:

[0007] Mixed PVB and water-soluble salt template: thermoplastic tough polymer (such as PVB), rigid filler (such as nano-SiO 2 ) and a micron-sized porogen sacrificial template (such as water-soluble NaCl) are mixed according to a set volume ratio to obtain a uniform mixed powder. The key to this step is to control the PVB, SiO 2 and NaCl, thereby affecting the porosity, pore size, rigidity and structural stability of the porous channels, thereby optimizing the delivery efficiency.

[0008] Preparing a precursor channel: putting the mixed powder into a mold, heating and melting the mixed powder to solidify the mixed powder, and cooling the mixed powder to obtain a precursor channel;

[0009] Removing the water-soluble salt template: Soaking the precursor channel in deionized water to remove the water-soluble salt template and obtain a porous channel structure. The precursor channel obtained in this step has good rigidity and stability, which is convenient for the operation of subsequent steps.

[0010] Encapsulation and syringe connection: The porous channel precursor and the syringe are encapsulated with a heat shrink tube to obtain a PVB material porous channel device for intracellular delivery. The device can be connected to a syringe to perform fluid extrusion operations, which is convenient for delivering various substances.

[0011] The porous channel device of the present invention has a simple structure, an easy-to-operate preparation process, excellent mechanical properties and biocompatibility, and can be widely used in the delivery of substances such as nucleic acids, proteins, and drugs in cells.

[0012] One of the technical problems solved by the present invention is the insufficiency of the existing intracellular delivery device in terms of cell delivery efficiency under high flux. In the existing technology, although there are a variety of cell delivery methods, such as electroporation, chemical transfection, etc., these methods either cause great damage to the cells, or have low flux and low delivery efficiency, resulting in the failure of the target substance to quickly and effectively enter the cells. In order to overcome these problems, the present invention provides a new porous channel device and a preparation method thereof, which optimizes the pore structure and uses mechanical stress to induce reversible deformation of the cell membrane, thereby promoting the smooth entry of the substance into the cell, and compared with the electroporation method and the chemical transfection method, it can effectively reduce cell damage. The present invention improves the flux by controlling the pore size and porosity of the porous channel, and uses mechanical stress to reduce damage to the cell while ensuring efficient delivery of exogenous substances. The pore size range of the porous channel is 5-15μm, which can effectively induce mechanical stress in the cell membrane, causing it to temporarily produce holes, thereby allowing the target substance to enter the cell. At the same time, the present invention adopts a solid-solid mixed template and a structure of precisely controlling the porous channel by adjusting the ratio of polymer, filler and water-soluble salt template and the curing process, thereby enhancing the connectivity, rigidity and stability of the porous channel and optimizing the cell delivery efficiency. In addition, the porous channel device of the present invention also has good adaptability, and the pore structure can be adjusted according to the needs of different types of cells to ensure the delivery efficiency of different substances. Compared with other delivery devices in the prior art, the method of the present invention is simple, low cost, and can achieve ideal results in the application of various cell types and delivery substances. The porous channel device of the present invention not only improves the cell delivery efficiency, but also effectively avoids the low flux, low efficiency and high loss problems common in the prior art, and provides a new technical path with broad application prospects.

[0013] As a preferred solution:

[0014] A porous channel device for intracellular delivery as described above, wherein the porous channel in the device is made of a thermoplastic tough polymer (such as polyvinyl butyral (PVB) material), a rigid filler (such as nano-SiO 2 ) and a micron-sized porogen sacrificial template (such as water-soluble sodium chloride (NaCl)).

[0015] A porous channel device for intracellular delivery as described above, wherein the mixing ratio of the PVB material and the micron-sized NaCl porogen is 3:7 to 7:3, preferably 4:6, to ensure good porosity and appropriate channel pore size.

[0016] A porous channel device for intracellular delivery as described above, wherein the nano-SiO 2 The filler accounts for 1-10 wt %, preferably 5 wt %, to ensure good mechanical strength, rigidity and stability of the porous channel.

[0017] A porous channel device for intracellular delivery as described above, wherein the porous channel has a porosity of 50%-80%, preferably a porosity of 60%, a connectivity of 60%-80%, preferably a connectivity of 80%, and a pore size of 5-15 μm, preferably a pore size of 10 μm, to optimize the efficiency of cell passage.

[0018] A porous channel device for intracellular delivery as described above, wherein the porous channel is cylindrical with a cylinder diameter of 5-10 mm and a length of 50-500 mm, preferably a cylindrical channel with a diameter of 6 mm and a length of 100 mm, to meet different experimental requirements.

[0019] In the porous channel device for intracellular delivery as described above, the micron-sized NaCl porogen is synthesized by dropping a NaCl solution into anhydrous ethanol, and the particle size range of the NaCl porogen is preferably 10 μm to ensure that the synthesized pore size is suitable for the passage of cells.

[0020] In the porous channel device for intracellular delivery as described above, the specifications of the micron-sized NaCl porogen are as follows:

[0021] The particle size of the micron-sized NaCl porogen is 10 μm: 20 ml of 5 M NaCl solution is added dropwise to 100 ml of anhydrous ethanol, stirred for 10 minutes, the white precipitate is collected by centrifugation, and then vacuum-dried at 100°C for 3 hours. The NaCl porogen with a particle size of 10 μm is obtained by grinding with agate smoke waves.

[0022] A porous channel device for intracellular delivery as described above, wherein the PVB, SiO 2 The mixture of the porogen and NaCl is treated in a vacuum drying oven during hot melt curing, with a curing temperature of 165°C to 185°C and a treatment time of 3 to 12 hours, preferably a curing condition of 175°C and 6 hours, to obtain porous channels with high strength and good stability.

[0023] A porous channel device for intracellular delivery as described above, wherein the precursor of the porous channel is soaked in deionized water to remove the NaCl template, and a porous structure is obtained by drying, the soaking time is 1-3 days, and the water is replaced every 6 hours to ensure efficient removal of NaCl.

[0024] As described above, a porous channel device for intracellular delivery is connected and packaged with a syringe via a heat shrink tube, and the heat shrink temperature is 115°C to 125°C, preferably 120°C, to ensure the stability and effectiveness of the device.

[0025] The method described above has the following specific steps:

[0026] (1) PVB material, nano-SiO 2 Mix with a micron-sized NaCl porogen in a set mass ratio and volume ratio (1-10wt%, preferably 5wt%; 3:7 to 7:3, preferably 6:4) to obtain a mixture. Put the mixture into a mold and perform hot melt curing by heating treatment. The curing temperature is 165°C to 185°C and the treatment time is 3 to 12 hours, preferably 175°C and 6 hours. After the curing is completed, demolding is performed to obtain a precursor channel;

[0027] (2) Soaking the precursor channel in deionized water for 1 to 3 days, changing the water every 6 hours to ensure effective removal of the NaCl porogen. After sufficient soaking, drying the porous channel precursor at a temperature of 20° C. to 40° C., preferably at 30° C. for 12 hours, in a vacuum drying oven;

[0028] (3) Connecting and encapsulating the obtained porous channel precursor to the syringe. Use a heat shrink tube to connect the precursor channel to the syringe mouth and perform heat shrink treatment at a temperature of 115°C to 125°C to ensure that the channel is stable and well sealed, thereby forming a porous channel device for intracellular delivery;

[0029] (4) in the final porous channel device, the pore size of the porous channel is ensured to be 5 to 15 μm, preferably 10 μm, the porosity is between 50% and 80%, preferably 60%, and the connectivity is 60%-80%, preferably 80%, so as to ensure that cells can pass smoothly and receive effective delivery;

[0030] (5) If necessary, the size and length of the porous channel can be adjusted according to different experimental requirements. The preferred channel size is 6 mm in diameter and 100 mm in length to ensure that the device can adapt to different types of cells and material delivery.

[0031] Beneficial effects:

[0032] (1) The porous channel device with high toughness and high rigidity for intracellular delivery of therapeutic cells of the present invention ensures that multiple cells can pass smoothly by precisely designing the pore size and porosity, and effectively promotes the delivery of substances by inducing mechanical stress. This structure significantly improves the delivery efficiency, has the advantage of high throughput, and avoids the problems of blockage or low throughput that may be caused by traditional single-cell operation methods;

[0033] (2) The preparation method of the present invention uses high molecular polymers such as PVB, rigid fillers such as nano-SiO 2and micron-scale sacrificial template porogens such as water-soluble sodium chloride, with pore sizes ranging from 5-15μm, 50%-80% porosity, and 60%-80% connectivity, good toughness, and impact strength greater than 20kJ / m 2 , high elongation at break of about 100%, strong rigidity, elastic modulus of about 50GPa, high mechanical strength, ensuring the ideal pore structure and stability of the porous channel, thereby achieving more efficient and stable substance delivery;

[0034] (3) The porous channel device for intracellular delivery of therapeutic cells of the present invention has high rigidity and stability. By using nano-SiO 2 (Silicon dioxide) fillers improve the rigidity of the porous channels, enhance the mechanical strength of the porous channels, optimize the service life and stability of the channels, and make them suitable for efficient delivery of different cell types (greater than 80%);

[0035] (4) The porous channel device for intracellular delivery of therapeutic cells of the present invention adopts a solid powder mixing process to ensure a high connectivity rate of the device, preferably 80%. This makes the porous channel have the advantage of high throughput, avoids the channel clogging problem commonly seen in traditional devices, and can efficiently deliver a large amount of substances into cells in a short time.

[0036] (5) The porous channel device for intracellular delivery of therapeutic cells of the present invention adopts a combination of PVB material and water-soluble salt template, has good biocompatibility, and can be widely used in the biomedical field, especially in high-throughput screening experiments such as gene transfection and drug delivery, and has excellent adaptability and high-throughput delivery capability;

[0037] (6) The porous channel device of the present invention is connected to the syringe by packaging with a heat shrink tube, which is easy to operate and ensures the effectiveness of the device in the experiment. In particular, it has high applicability in cell delivery and can meet various experimental requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a SEM image of the cross-section of the PVB porous channel precursor prepared in Example 1.

[0039] Figure 2 This is a physical picture of the porous channel device prepared in Example 1 for intracellular delivery of therapeutic cells.

[0040] Figure 3 The flow cytometric graphs of the delivery efficiency and cell viability of the porous channel device prepared in Example 1 are shown. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0042] Example 1

[0043] A preparation method of a porous channel device for intracellular delivery, the steps of which are mainly as follows:

[0044] (1) First, synthesize a micron-sized NaCl water-soluble pore-forming agent. Take 20 ml of a 5 M NaCl solution (the solvent is water), and dropwise add it to 100 ml of absolute ethanol under vigorous stirring. Stir and react for 10 minutes, stop stirring, and let it stand for 1 minute. Pour off the supernatant, and centrifuge at a speed of 6000 revolutions per minute for 5 minutes to collect the white precipitate. Wash the precipitate with absolute ethanol, centrifuge again, and collect the precipitate. Place the collected precipitate in a vacuum drying oven at 100 °C and dry it for 3 hours. After taking it out, grind it into powder with an agate mortar to finally obtain a micron-sized NaCl pore-forming agent with a particle size of 10 μm.

[0045] (2) Mix PVB (polyvinyl butyral), the synthesized NaCl water-soluble pore-forming agent, and nano-SiO 2 (silicon dioxide) according to a set volume ratio (the volume ratio of PVB to NaCl is 4:6, and the addition amount of nano-SiO 2 is 5 wt%) to obtain a uniform mixed powder. Grind this mixture with an agate mortar for 10 minutes until it is completely uniform;

[0046] (3) Load this mixed powder into a mold and perform hot melt curing treatment at a temperature of 175 °C for 6 hours. After the treatment is completed, let it cool naturally and demold to obtain a precursor channel;

[0047] (4) Immerse the precursor channel in deionized water to remove the water-soluble NaCl pore-forming agent template. The immersion time is 2 days, and the water is changed every 6 hours. After desalting is completed, take out the precursor channel and dry it in a vacuum drying oven at 30 °C for 12 hours to obtain a porous channel precursor;

[0048] (5) Enclose the porous channel precursor in a syringe through a heat-shrinkable tube to ensure a stable connection between the porous channel and the syringe. The heat shrinkage temperature is controlled between 115 °C and 125 °C, and a stable porous channel device is formed after completion.

[0049] Finally, a porous channel device for intracellular delivery was prepared, with a pore size of about 10 μm, a porosity of 60%, and a connectivity of 80%. The device can efficiently deliver substances into cells while avoiding the problems of channel blockage or low flux in traditional operations. The SEM image of the cross-section of the prepared PVB porous channel precursor is shown below: Figure 1 As shown; the actual picture of the porous channel device for intracellular delivery is as shown Figure 2 As shown; the flow cytometric graphs of the porous channel device delivery efficiency and cell viability are shown Figure 3 shown.

[0050] This embodiment ensures the ideal pore structure, high connectivity and high stability of the porous channel by accurately designing the mixing ratio, porosity and pore size of PVB and NaCl porogen, and effectively promotes the delivery of substances. The preparation process is simple and the cost is low. The materials used (such as PVB, SiO 2 and NaCl) are readily available and suitable for industrial production. The device has a high throughput and can deliver a large amount of substances into cells in a short time, and is suitable for applications such as high-throughput screening. The pore size and porosity of the device can be adjusted according to different cell types and delivery requirements, and has strong adaptability; the design of the porous channel device improves the efficiency of cell delivery, can reduce the clogging problem in traditional methods, and ensure the smooth progress of the experiment.

[0051] The device has high rigidity, which is due to the nano-SiO 2 (Silicon dioxide) filler addition. Nano-SiO 2 The addition of significantly improves the rigidity and stability of the porous channel, thereby enhancing the mechanical strength of the channel. It not only optimizes the service life and stability of the porous channel, but also enables it to withstand greater physical stress and avoid deformation or rupture during high-throughput delivery. In addition, higher rigidity also improves the device's adaptability to cells during delivery, ensuring that it can work stably under various cell types and different experimental conditions, further improving the efficiency and accuracy of cell delivery.

[0052] The device has high connectivity, which is mainly due to its solid powder mixing process and optimized pore structure design. 2The mixing ratio of the water-soluble salt template and the grinding process ensure the high connectivity of the pores in the porous channel. The connectivity rate is as high as 80%, which means that enough interconnected channels are formed in the channel, which greatly improves the flux and efficiency of substance delivery. High connectivity not only reduces the risk of channel blockage, but also ensures that the substances in the delivery process can pass through the channel quickly and evenly, improving the fluency and operability of the experiment. Through this design, the device can achieve effective delivery of a large amount of substances in a relatively short time, which is particularly suitable for application scenarios such as high-throughput screening and large-scale cell delivery.

[0053] Comparative Example 1

[0054] A method for preparing a porous channel device for intracellular delivery, wherein the steps are substantially the same as those of Example 1, except that the volume ratio of PVB to NaCl water-soluble porogen in step (2) is 5:5. After mixing using this volume ratio, the porous channel device obtained has a pore size of 10 μm and a porosity of 50%. Compared with Example 1, the final delivery effect has a slightly lower flux, and the pressure during operation is relatively high, which is prone to slight clogging problems.

[0055] Comparative Example 2

[0056] A method for preparing a porous channel device for intracellular delivery, wherein the steps are substantially the same as those of Example 1, except that the particle size of the NaCl water-soluble porogen mixed in step (2) is 5 μm. The porous channel device prepared with this particle size has a smaller pore size of 6 μm and a porosity of 60%. Although the device has a higher porosity, due to the smaller pore size, cells are squeezed into the channel, the pressure is higher during operation, and the clogging problem is serious.

[0057] Comparative Example 3

[0058] A method for preparing a porous channel device for intracellular delivery, wherein the steps are substantially the same as those in Example 1, except that the step (2) of omitting the nano-SiO 2 The rigidity of the porous channel device is reduced, and the mechanical strength of the channel is relatively weak. In addition, the stability and pore structure of the channel may be less than ideal, which may lead to lower delivery efficiency and flux, and there may be certain clogging problems in high-throughput applications.

[0059] Comparative Example 4

[0060] A method for preparing a porous channel device for intracellular delivery, wherein the steps are substantially the same as those of Example 1, except that the curing temperature in step (3) is 160° C. and the curing time is 5 hours. Since the curing temperature is too low, the mechanical strength and integrity of the porous channel device finally obtained are poor, and deformation and breakage easily occur during use, resulting in poor stability of substance delivery.

[0061] Comparative Example 5

[0062] A preparation method of a porous channel device for intracellular delivery, the steps of which are basically the same as those in Example 1, except that the deionized water soaking treatment in step (4) is omitted or the soaking is not thorough, and drying is directly carried out. Due to the incomplete removal of the NaCl porogen template, there is more residual NaCl in the finally prepared porous channel device, resulting in a smaller pore size and non - flow of the porous channels, and the substance cannot be effectively delivered.

[0063] Example 2

[0064] A preparation method of a porous channel device for intracellular delivery, the steps of which are mainly as follows:

[0065] (1) First, synthesize a micron - sized NaCl water - soluble porogen: Take 20 ml of a 4M NaCl solution (the solvent is water), and dropwise add it to 150 ml of absolute ethanol under vigorous stirring conditions. Stir and react for 5 minutes, stop stirring, and let it stand for 2 minutes. Pour off the supernatant, use a centrifuge to centrifuge at a speed of 8000 revolutions per minute for 10 minutes, and collect the white precipitate. Wash the precipitate with absolute ethanol, centrifuge again with a centrifuge, and collect the precipitate. Place the collected precipitate in a vacuum drying oven at 80 °C and dry for 5 hours. After taking it out, grind it into powder with an agate mortar, and finally obtain a micron - sized NaCl porogen with a particle size of 8 μm;

[0066] (2) Mix PVB (polyvinyl butyral), the synthesized NaCl water - soluble porogen, and nano - SiO 2 (silicon dioxide) according to a set volume ratio (the volume ratio of PVB to NaCl is 3:7, and the addition amount of nano - SiO 2 is 10 wt%) to obtain a uniform mixed powder. Grind this mixture with an agate mortar for 15 minutes until it is completely uniform;

[0067] (3) Load the mixed powder into a mold and carry out hot - melt curing treatment at a temperature of 180 °C for 8 hours. After the treatment is completed, cool it naturally and demold to obtain a precursor channel;

[0068] (4) Immerse the precursor channel in deionized water to remove the water - soluble NaCl porogen template. The soaking time is 3 days, and the water is changed every 4 hours. After desalting is completed, take out the precursor channel and dry it in a vacuum drying oven at 40 °C for 24 hours to obtain a porous channel precursor;

[0069] (5) Enclose the porous channel precursor into a syringe through a heat - shrinkable tube to ensure a stable connection between the porous channel and the syringe. The heat - shrink temperature is controlled between 120 °C and 130 °C, and a stable porous channel device is formed after completion.

[0070] Finally, a porous channel device for intracellular delivery is prepared. The pore size of the device is about 8 μm, the porosity is 65%, and the connectivity is 70%. This device can efficiently deliver substances into cells while avoiding problems such as channel blockage or low flux in traditional operations.

[0071] In this example, by adjusting the mixing ratio of PVB, SiO 2 and the NaCl porogen and the hot melt treatment conditions, the prepared porous channel device has good mechanical strength and appropriate pore size distribution, and can more efficiently meet the needs of cell delivery; the preparation process is simple and the cost is low, which is suitable for large-scale industrial production; compared with Example 1, the NaCl porogen used in this example has a smaller particle size and a more uniform pore size distribution, which is suitable for the experimental needs of fine cell delivery and has a wider applicability.

[0072] Example 3

[0073] A preparation method of a porous channel device for intracellular delivery, the steps of which are mainly as follows:

[0074] (1) First, synthesize a micron-sized NaCl water-soluble porogen: Take 15 ml of a 5 M NaCl solution (the solvent is water), and dropwise add it to 120 ml of absolute ethanol under vigorous stirring. Stir and react for 8 minutes, stop stirring, and let it stand for 2 minutes. Pour off the supernatant, and centrifuge at a speed of 7000 revolutions per minute for 8 minutes to collect the white precipitate. Wash the precipitate with absolute ethanol, centrifuge again with a centrifuge, and collect the precipitate. Place the collected precipitate in a vacuum drying oven at 90 °C and dry for 4 hours. After taking it out, grind it into powder with an agate mortar and pestle, and finally obtain a micron-sized NaCl porogen with a particle size of 9 μm;

[0075] (2) Mix PVB (polyvinyl butyral) with the synthesized NaCl water-soluble porogen and nano-SiO 2 (silicon dioxide) according to a set volume ratio (the volume ratio of PVB to NaCl is 5:5, and the addition amount of nano-SiO 2 is 5 wt%) to obtain a uniform mixed powder. Grind this mixture with an agate mortar and pestle for 15 minutes until it is completely uniform;

[0076] (3) Load the mixed powder into a mold and perform hot melt curing treatment at a temperature of 170 °C for 7 hours. After the treatment is completed, cool it naturally and demold it to obtain a precursor channel;

[0077] (4) Immerse the precursor channel in deionized water to remove the NaCl porogen template for 2 days, and change the water every 4 hours. After desalting is completed, take out the precursor channel and dry it in a vacuum drying oven at 35 °C for 14 hours to obtain a porous channel precursor;

[0078] (5) Encapsulate the porous channel precursor into the syringe through a heat shrink tube to ensure that the porous channel is firmly connected to the syringe. The heat shrink temperature is controlled between 120°C and 125°C to form a stable porous channel device.

[0079] The porous channel device for intracellular delivery was finally prepared, with a pore size of about 9 μm, a porosity of 65%, and a connectivity of 70%. The device can efficiently deliver substances into cells while avoiding the problems of blockage or low flux in traditional methods.

[0080] In this embodiment, by optimizing the mixing ratio of PVB and NaCl porogen and the hot melt curing temperature, the porous channel device prepared has significantly improved structural integrity, delivery efficiency and adaptability; it is suitable for medium-scale high-throughput cell experiments, and has certain cost advantages, which is convenient for industrial application.

[0081] Example 4

[0082] A method for preparing a porous channel device for intracellular delivery, the steps of which are mainly:

[0083] (1) Synthesis of micron-sized NaCl water-soluble porogen: Take 10 ml of 4M NaCl solution (the solvent is water), add it dropwise into 150 ml of anhydrous ethanol under vigorous stirring conditions, stir and react for 6 minutes, stop stirring, and let it stand for 2 minutes. Pour off the supernatant, use a centrifuge to centrifuge at 6000 rpm for 6 minutes, and collect the white precipitate. Wash the precipitate with anhydrous ethanol, repeat the centrifugation once, and collect the precipitate. Place the collected precipitate in a vacuum drying oven at 80°C and dry it for 5 hours. After taking it out, use agate smoke wave to grind it into powder, and finally obtain a micron-sized NaCl porogen with a particle size of 7μm;

[0084] (2) PVB (polyvinyl butyral) was mixed with a synthesized NaCl water-soluble porogen and nano-SiO 2 (Silicon dioxide) according to the set volume ratio (the volume ratio of PVB to NaCl is 4:6, nano-SiO 2 The mixture was ground using an agate mortar for 15 minutes until it was completely uniform;

[0085] (3) The mixed powder is placed in a mold and subjected to hot melt curing treatment at a temperature of 180° C. for 5 hours. After cooling and demolding, a precursor channel is obtained;

[0086] (4) Soaking the precursor channel in deionized water to remove the NaCl porogen template for 3 days, changing the water every 6 hours. After desalting, the precursor channel was taken out and dried in a vacuum drying oven at 40°C for 16 hours to obtain a porous channel precursor;

[0087] (5) Encapsulate the porous channel precursor into the syringe through a heat shrink tube to ensure that the porous channel is firmly connected to the syringe. The heat shrink temperature is controlled between 125°C and 130°C to form a stable porous channel device.

[0088] Finally, a porous channel device for intracellular delivery was prepared, with a pore size of about 7 μm, a porosity of 70%, and a connectivity of 65%. The device is suitable for the efficient delivery of small particles and has the characteristics of high flux and low clogging rate.

[0089] This embodiment significantly improves the throughput of the device by controlling the pore size and optimizing the solidification temperature, and is suitable for the high-precision delivery requirements of special cell types.

[0090] Example 5

[0091] A method for preparing a porous channel device for intracellular delivery, the steps of which are mainly:

[0092] (1) Synthesis of micron-sized NaCl water-soluble porogen: Take 25 ml of 5M NaCl solution (solvent is water), add it dropwise into 150 ml of anhydrous ethanol under vigorous stirring conditions, stir and react for 7 minutes, stop stirring, and let it stand for 1 minute. Pour off the supernatant, use a centrifuge to centrifuge at 7500 rpm for 8 minutes, and collect the white precipitate. Wash the precipitate with anhydrous ethanol, centrifuge again, and collect the precipitate. Place the precipitate in a vacuum drying oven at 85°C and dry it for 6 hours. After taking it out, grind it into powder with agate smoke wave, and finally obtain a micron-sized NaCl porogen with a particle size of 10μm;

[0093] (2) PVB (polyvinyl butyral) was mixed with a synthesized NaCl water-soluble porogen and nano-SiO 2 (Silicon dioxide) according to the set volume ratio (the volume ratio of PVB to NaCl is 5:5, nano-SiO 2 The mixture was ground using an agate mortar for 15 minutes until it was completely uniform;

[0094] (3) The mixed powder is placed in a mold and subjected to hot melt curing treatment at a temperature of 185° C. for 6 hours. After the treatment is completed, the mold is cooled and demolded to obtain a precursor channel;

[0095] (4) Soaking the precursor channel in deionized water to remove the NaCl porogen template for 2 days, changing the water every 4 hours. After desalting, the precursor channel was taken out and dried in a vacuum drying oven at 30°C for 10 hours to obtain a porous channel precursor;

[0096] (5) The porous channel precursor is encapsulated into the syringe through a heat shrink tube to ensure that the porous channel is firmly connected to the syringe. The heat shrinking temperature is controlled between 120°C and 125°C, and a stable porous channel device is formed after completion.

[0097] Finally, a porous channel device for intracellular delivery was prepared, with a pore size of 10 μm, a porosity of 60%, and a connectivity of 80%. The device can stably and efficiently deliver substances into cells and is suitable for industrial production.

[0098] This embodiment optimizes the pore structure and mechanical strength of the device by reasonably adjusting the mixing ratio and hot melt curing temperature. It is suitable for application scenarios such as high-throughput screening and gene delivery, and has excellent adaptability and reliability.

Claims

1. A porous channel device with high toughness and high rigidity for intracellular delivery of therapeutic cells and a method for preparing the same, which allows cells to pass through and induces mechanical stress on the cells, thereby promoting the introduction of one or more substances into the cells, characterized in that The following steps are involved: A thermoplastic tough polymer (such as polyvinyl butyral (PVB), polycarbonate (PC), polypropylene (PP), etc.) is mixed with a rigid filler (such as nano silicon dioxide (SiO2), nano calcium carbonate (CaCO3), nano zinc oxide (ZnO2), etc.) and a micron-sized porogen sacrificial template (such as water-soluble sodium chloride (NaCl), sucrose, calcium carbonate, etc.) according to a set volume ratio to obtain a mixed powder; The powder is placed in a mold for hot melting and solidification, and a precursor channel is obtained after demoulding; Soaking the precursor channel in water to remove the water-soluble salt template and then drying to obtain a porous channel precursor; The porous channel precursor is then packaged with a syringe to obtain a porous channel device for intracellular delivery.

2. The porous channel according to claim 1, characterized in that The porous channel is a cylinder with a diameter of 5-10 mm and a length of 50-500 mm. The volume ratio of the mixed materials is set to control the porosity to 50%-80%. The solid-solid mixing process is used to achieve a connectivity rate of 60%-90% and a pore size of 5-15 μm.

3. The method for synthesizing porous channels using a water-soluble salt template method according to claim 1, characterized in that: Thermoplastic tough polymers such as polyvinyl butyral (PVB) have good toughness and an impact strength of about 20-40 kJ / m 2 , fracture toughness up to 150kJ / m 2 , high elongation at break is 100%-300%.

4. The method for synthesizing porous channels by water-soluble salt template method according to claim 1, characterized in that: The rigid filler is nano-silicon dioxide (SiO2), with an elastic modulus of about 70-120 GPa, a hardness of up to 1000 HV, and a particle size of 10-50 nm; the rigid filler is nano-SiO2, and its addition amount is 1-10wt%, which can improve the rigidity of the channel, and the elastic modulus is about 50 GPa.

5. The method for synthesizing porous channels using a water-soluble salt template method according to claim 1, characterized in that: The water-soluble salt template is sodium chloride, and micron-sized sodium chloride particles are prepared in advance with a particle size of 5-15 μm; the volume ratio of the thermoplastic tough polymer PVB to the water-soluble salt template is 3-7:3-7 to ensure the best pore structure and stability.

6. The method for synthesizing porous channels using a water-soluble salt template method according to claim 1, characterized in that: The curing is carried out under heating conditions, using a vacuum drying oven, with a curing temperature of 165-185°C and a processing time of 3-12 hours; after the hot melt curing treatment is completed, the precursor channel is obtained naturally after demoulding.

7. The method for synthesizing porous channels using a water-soluble salt template method according to claim 1, characterized in that: The precursor channel is soaked in deionized water to remove the water-soluble sodium chloride. The soaking time is 1-3 days, the water is changed every 6 hours, and then dried to obtain a porous channel precursor.

8. The method for preparing a porous channel device for intracellular delivery according to claim 1, characterized in that: The porous channel precursor is connected and packaged with a heat shrink tube, and the heat shrink temperature is 115-125°C.

9. A porous channel device for intracellular delivery, characterized in that: The porous channel is prepared by the method of synthesizing porous channels by the water-soluble salt template method according to any one of claims 1 to 8, with a porosity of 50% to 80%, a pore size of 5 to 15 μm, and an impact strength of more than 20 kJ / m 2 , high elongation at break is about 100%, elastic modulus is about 50GPa, and mechanical strength is high.

10. The method for preparing a porous channel device for intracellular delivery according to claim 1, characterized in that: When used for intracellular delivery of functional therapeutic cells, human T lymphocyte leukemia cells (Jurkat) were selected as the functional therapeutic cell model, and FITC-Dextran 10 was used as the model molecule for delivery. The efficiency of delivering FITC-Dextran 10 into Jurkat cells can reach up to 84.3%, the cell survival rate can reach up to 98.2%, and the net acquisition rate can reach 82.9%.

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