High-porosity and high-connectivity porous medium material for intracellular delivery of therapeutic cells and preparation method of porous medium material

Abstract

The invention relates to a high-porosity and high-connectivity porous medium material for intracellular delivery of therapeutic cells and a preparation method of the high-porosity and high-connectivity porous medium material. According to the microchannel, a porous polymer medium is prepared in a hard pipeline through a hard template method, and the specific preparation method comprises the following steps: stirring and mixing a thermosetting polymer and alcohol-soluble particles, performing high-temperature curing, removing the alcohol-soluble particles through absolute ethyl alcohol, forming a porous structure, and constructing the microchannel for intracellular delivery of animal cells. The channel with the porous medium material has excellent conductivity, and the connectivity between pore diameters can reach 98%. And the pore size distribution is wide and is between 5 microns and 50 microns. On the premise of effectively enhancing the permeability of cell membranes, good cell activity is ensured, efficient and safe intracellular delivery is realized, the highest delivery efficiency can reach 91.2%, the highest cell survival rate can reach 97.2%, and the net yield can reach 88.6%. Meanwhile, the method is simple in preparation process, convenient to operate and particularly suitable for the fields of gene therapy, drug development, cell research and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of intracellular cell delivery, and relates to the construction of an integrated delivery platform based on micron-scale porous medium materials and its application in the intracellular delivery of functional therapeutic cells. Background Art

[0002] With the rapid development of biomedicine and genetic engineering, the research and application of macromolecule delivery in animal cells is becoming a bridge between basic research and clinical treatment. Animal intracellular delivery is the process of delivering exogenous macromolecules (such as genes, drugs or proteins) into the interior of cells. This has a wide range of applications in the biomedical field, especially in gene therapy, drug development and cell biology research. An effective delivery system can significantly improve the delivery efficiency of drugs and genes and reduce side effects, providing new hope for the treatment of many intractable diseases. In the future, with the continuous improvement of delivery technology, animal cell intracellular delivery is expected to play a more important role in personalized medicine, gene editing and disease treatment.

[0003] At present, there are two types of methods for intracellular delivery of animal cells: carrier-based and membrane-disruption-based. Compared with the limitations of carrier-mediated delivery methods on specific cell types and the cell damage caused by delivery methods based on chemical membrane disruption, physical membrane disruption delivery methods have emerged as a promising alternative. Traditional physical membrane disruption delivery methods, such as microneedles and electroporation, are often used to deliver biomolecules into cells. However, these methods often have difficulty in achieving high levels of intracellular delivery, cell survival, and delivery efficiency at the same time. It is worth noting that based on the tiny size of the channel and the ability to control microfluidics, the emerging microfluidic chip technology can achieve precise control of cells and realize efficient and safe cell delivery processes. However, the cell delivery system based on microfluidic chips also has shortcomings. The channel is easily blocked, resulting in a decrease in flux, and the preparation process is difficult and expensive. Therefore, finding a high-throughput, efficient and safe intracellular delivery method with a simple preparation process and low cost remains one of the focuses of current research. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems existing in the prior art and to provide an integrated delivery platform based on micron-scale porous medium materials, which has a simple preparation process and low cost and can achieve high-throughput, efficient and safe delivery of functional therapeutic cells.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] An integrated delivery platform based on micron-scale porous media materials is constructed. A three-dimensional porous media material with a wide micron-scale pore size distribution and good connectivity is prepared by a hard template method. Through the synergistic principle of fluid shear and cell extrusion, exogenous functional macromolecules are efficiently and safely delivered into cells. By applying the integrated delivery platform based on porous structure microchannels to intracellular delivery, high-throughput, non-clogging cell delivery can be achieved;

[0007] One of the technical problems solved by the present invention is that the narrow microchannels of traditional microfluidic chips are prone to blockage during delivery due to the precise control of cells; the present invention successfully prepares a porous medium and assembles it into an integrated delivery platform. The porous medium is prepared by a hard template method, and the alcohol-soluble particles and thermosetting polymers are mixed and transferred to a hard pipe for solidification. The alcohol-soluble particles in the hard pipe are dissolved with anhydrous ethanol to form a porous medium microchannel. The microchannel, adapter, and syringe are assembled, and the sample solution in the syringe is pushed by a propulsion pump to complete the delivery process through the microchannel. The diameter of the hard pipe is millimeter-level, which greatly increases the processing area of ​​the cells during the delivery process, thereby reducing the blockage during the delivery process. It should be noted that when the density of the delivered cells is extremely large, the diameter of the microchannel can also be increased accordingly to increase the processing area of ​​the delivery and reduce the risk of blockage. The present invention regulates the size of the microchannel pore size and the porosity, so that the microchannel not only ensures the destruction of the cell membrane during the functional therapeutic cell delivery process, but also avoids the blockage caused by multiple cells passing through at the same time. The hard template used in the preparation of porous medium materials is citric acid monohydrate. The size of citric acid monohydrate powder can be adjusted by adjusting the grinding time of the grinder, thereby adjusting the size of the pores of the porous medium material. In addition, the polymer used in the preparation is Polydimethylsiloxane (PDMS), which is liquid at room temperature and can be solidified at high temperature. By adjusting the mixing ratio of PDMS and citric acid monohydrate powder at room temperature, the porosity of the porous medium material can be adjusted. In addition, many existing microfluidic chip delivery technologies are single-mechanism membrane ruptures, such as cell extrusion, puncture, fluid shear, etc., and the present invention provides a new idea. Since the pore size of the ground citric acid monohydrate is not completely consistent, the prepared porous medium material has a wide pore size distribution, so that the cell delivery process is coordinated by two different delivery mechanisms. For pores larger than the cell diameter, the cell membrane is mainly destroyed by fluid shear to increase permeability; for pores smaller than the cell diameter, the cell membrane is mainly destroyed by the squeezing force between the cell and the pore, thereby delivering. The synergistic effect of the two delivery methods further improves the delivery efficiency and flux.

[0008] As a preferred solution:

[0009] In the porous medium material-mediated intracellular delivery of functional therapeutic cells as described above, the prepared porous medium material hard template is citric acid monohydrate, the medium material is PDMS, and the microchannel is encapsulated by a silicone tube.

[0010] As described above, in the porous medium material-mediated intracellular delivery of functional therapeutic cells, the prepared porous medium material hard template is citric acid monohydrate, the medium material is PDMS, and the microchannel is encapsulated by a silicone tube. In theory, sodium chloride and sucrose can also be used as the hard template, but considering the good solubility in anhydrous ethanol and the non-reaction with the solution, citric acid monohydrate was selected as the hard template. At the same time, it is required that it can be mixed evenly with the hard template and injected, and it can maintain good mechanical properties. PDMS, which is liquid at room temperature and can be cured at high temperature, is selected as the medium material. At the same time, in order to ensure good adhesion with the porous medium, silicone tubes are selected as the encapsulation of the porous medium material.

[0011] In the above-mentioned porous medium material-mediated intracellular delivery of functional therapeutic cells, the particle size of the citric acid monohydrate particles is 0.15 to 0.25 microns; too small a particle size will cause the cells to be squeezed and blocked during the delivery process, and too large a particle size will cause the interaction between the cells and the microchannel to be reduced during the delivery process, resulting in low delivery efficiency;

[0012] In the above-mentioned porous medium material-mediated intracellular delivery of functional therapeutic cells, the volume ratio of PDMS to micron particles is 40%. A too small volume ratio may easily lead to poor dissolution of citric acid monowater and poor microchannel connectivity; a too large volume ratio may easily lead to reduced mechanical strength and rigidity of the microchannel.

[0013] The porous medium material-mediated intracellular delivery of functional therapeutic cells as described above, wherein a vertical propulsion pump is used to propel the syringe to complete the delivery process. A horizontal propulsion pump may cause the deposition of Jurkat cells to a certain extent, and the use of a vertical propulsion pump avoids this problem;

[0014] The method described above has the following specific steps:

[0015] (1) Grind citric acid monohydrate particles with a grinder for 30 seconds to 2 minutes until they are powdered, weigh PDMS and put it into a vacuum oven, evacuate at room temperature for 5 minutes to remove bubbles, add citric acid monohydrate powder at a volume ratio of 25% to 40%, stir with a mixer for 10 minutes until the mixture is uniform, and then evacuate in a vacuum oven for 10 minutes to remove bubbles;

[0016] (2) The vacuumed solution is injected into a silicone tube with a syringe. The silicone tube has an outer diameter of 5 mm, an inner diameter of 3 mm, and a length of 1 to 4 cm. The injection mixture is about 0.5 to 3 cm. The silicone tube containing the mixed solution is placed in an oven at 80 degrees and cured at high temperature for 2 hours.

[0017] (3) Immersing the solidified microchannel in 60-degree anhydrous ethanol to dissolve citric acid monohydrate. The heated anhydrous ethanol accelerates the dissolution of citric acid monohydrate. The dissolution is performed for 6 hours. After the dissolved microchannel is drained of the remaining anhydrous ethanol, it is placed in an oven and dried at 80 degrees for 1 hour.

[0018] (4) Assembling the prepared microchannel, adapter, and syringe, and advancing the syringe using a vertical push pump, adjusting the flow rate to 10-100 mL / h, so that the solution in the syringe completes the delivery process through the microchannel;

[0019] (5) The delivered cells were collected in a centrifuge tube, live-death dyes were added, and the delivery efficiency and live-death rate were analyzed using flow cytometry and confocal laser microscopy;

[0020] Beneficial effects:

[0021] (1) The present invention is an integrated system-mediated intracellular delivery of functional therapeutic cells based on porous medium materials. The porous medium materials are assembled into an integrated delivery system, which maintains the sterility and speed of the delivery process. The good connectivity of the channel itself and the increase in the cross-section of the microchannel can reduce the blockage during cell delivery and improve the delivery flux.

[0022] (2) The present invention is an integrated system based on porous media materials for intracellular delivery of functional therapeutic cells. The microchannel itself has a wide pore size distribution range. A pore size of 10-20 microns can destroy the cell membrane by squeezing the cell, and a pore size of 30-50 microns can destroy the cell membrane by fluid shearing. By controlling the pore size and porosity of the microchannel, the functional medical cell membrane is destroyed by synergistic cell squeezing and fluid shearing mechanisms, thereby improving the delivery efficiency;

[0023] (3) The porous medium material-mediated intracellular delivery of functional therapeutic cells of the present invention has low cost and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the SEM and pore distribution diagram of microchannel;

[0025] Figure 2 It is the microchannel μ-CT and porosity distribution map;

[0026] Figure 3 This is a confocal microscopy image of delivery efficiency and cell activity in specific example 2; DETAILED DESCRIPTION

[0027] 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 are not intended 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 fall within the scope limited by the appended claims of the application equally.

[0028] Example 1

[0029] A preparation method of a porous medium material comprises the following steps:

[0030] (1) Grind citric acid monohydrate particles into powder using a grinder for 1 min, weigh PDMS and put it into a vacuum oven, evacuate at room temperature for 5 min to remove bubbles, add citric acid monohydrate powder at a volume ratio of 35%, stir with a blender for 10 min until the mixture is uniform, and then evacuate in a vacuum oven for 10 min to remove bubbles;

[0031] (2) The vacuumed solution is injected into a silicone tube with a syringe. The silicone tube has an outer diameter of 5 mm, an inner diameter of 3 mm, and a length of 1 to 3 cm. The injection mixture is about 0.5 to 2 cm. The silicone tube containing the mixed solution is placed in an oven at 80 degrees and cured at high temperature for 2 hours.

[0032] (3) The cured microchannel is immersed in anhydrous ethanol at 60 degrees to dissolve citric acid monohydrate. The heated anhydrous ethanol accelerates the dissolution of citric acid monohydrate, and the dissolution is carried out for 6 hours. After the dissolved microchannel is drained of the remaining anhydrous ethanol, it is placed in an oven and dried at 80 degrees for 1 hour.

[0033] The final micron-porous structure-based animal cell intracellular delivery microchannel is a PDMS porous channel wrapped by a silicone tube, such as Figure 1 As shown, the PDMS in the tube is evenly dispersed with pores of different sizes. The pores are formed by citric acid monohydrate dissolved in anhydrous ethanol. The average pore size is 35.2 microns and the median pore size is 11.8 microns. Figure 2 As shown, the prepared porous material has good porosity and connectivity, and the porosity size is 39.5%;

[0034] Example 2

[0035] A porous medium material mediates intracellular delivery of functional therapeutic cells, the steps are as follows:

[0036] (1) Jurkat cells were removed from the incubator and centrifuged at 2000 rpm for 2 min; then resuspended in RPMI 1640 medium;

[0037] (2) Jurkat cells, 10 kDa FITC-dextran, and RPMI 1640 medium were prepared according to a 10 kDa FITC-dextran concentration of 0.5 mg / mL and a cell density of 1 million / mL;

[0038] (3) Use a pipette to add 200 μL of culture medium to a 1.5 mL syringe and assemble the syringe, adapter, and microchannel;

[0039] (4) The cells were delivered at a rate of 10 mL / h using a vertical propulsion pump. The delivered cells were collected in a 1.5 mL centrifuge tube, washed with PBS, and then analyzed using CellTrace TM Calcein orange-red, AM and TO-PRO-3 were used to stain the cells for live and dead, and then the treated cells were processed and analyzed by flow cytometry.

[0040] The cells finally delivered have higher cell activity and delivery efficiency. Figure 3 As shown, the delivery efficiency can reach up to 91.2%, the cell survival rate can reach up to 97.2%, and the net acquisition rate can reach 88.6%.

[0041] Comparative Example 1

[0042] A porous medium material mediated intracellular delivery of functional therapeutic cells, the steps of which are basically the same as those of Example 1 and Example 2, except that the flow rates in step (4) of Example 2 are 20, 50, and 100 mL / h, and the net cell acquisition rate after delivery is lower than that of Example 2. The flow rates are 20, 50, and 100 mL / h, and the net cell acquisition rates after delivery are 30.0%, 23.1%, and 16.2%, respectively.

[0043] Comparative Example 2

[0044] A porous medium material mediated intracellular delivery of functional therapeutic cells, the steps of which are basically the same as those of Example 1 and Example 2, except that in step (2) of Example 2, the cell density is 2 million, 4 million, and 8 million / mL, and the net cell acquisition rate after delivery is lower than that of Example 2. The cell density is 2 million, 4 million, and 8 million / mL, and the net cell acquisition rate after delivery is 62.1%, 61.1%, and 30.4%, respectively.

[0045] Comparative Example 3

[0046] A porous medium material-mediated intracellular delivery of functional therapeutic cells, the steps of which are substantially the same as those of Example 1 and Example 2, except that in step (2) of Example 1, the length of the silicone tube is 0.5, 1.5, and 2 cm, and the net cell acquisition rate after delivery is lower than that of Example 1. The length of the silicone tube is 0.5, 1.5, and 2 cm, and the net cell acquisition rate after delivery is 50.1%, 59.8%, and 68.2%, respectively.

Claims

1. A porous medium material-mediated intracellular delivery of functional therapeutic cells, characterized in that: An integrated delivery system based on porous microchannels was assembled, combining porous medium microchannels with intracellular delivery. The delivery microchannels have a wide pore size distribution and high connectivity, which can achieve efficient, safe, and high-throughput delivery effects. The assembled delivery system can achieve sterile and fast delivery process.

2. The microchannel for intracellular delivery of functional therapeutic cells according to claim 1, characterized in that: The microchannel is prepared using a hard template method, in which the template is an alcohol-soluble particle with a certain water absorption (such as citric acid monohydrate particles, tartaric acid, anhydrous magnesium sulfate, anhydrous calcium chloride, etc.), which does not react with the polymer and is easily soluble in anhydrous ethanol.

3. The microchannel for intracellular delivery of functional therapeutic cells according to claim 2, characterized in that: The particle size of the alcohol-soluble particles is controlled to be 0.15 to 0.25 microns, and they are easy to deliquesce and bond.

4. The microchannel for intracellular delivery of functional therapeutic cells according to claim 2, characterized in that: Thermosetting polymers are used as the main body of the porous medium, and the polymers have the property of transforming from liquid to solid at high temperature.

5. The microchannel for intracellular delivery of functional therapeutic cells according to claim 4, characterized in that: The polymer and the alcohol-soluble particles are stirred by a stirrer to form a uniform mixed solution.

6. The microchannel for intracellular delivery of functional therapeutic cells according to claim 5, characterized in that: Use a syringe to move the mixed solution into the hard pipe, injecting the mixed solution at a depth of 0.5 to 3 mm.

7. The microchannel for intracellular delivery of functional therapeutic cells according to claim 6, characterized in that: The silicone tube filled with the mixed liquid is placed in an oven for high-temperature curing. The tube filled with the mixed liquid is soaked in anhydrous ethanol to dissolve the alcohol-soluble particles inside and form a porous foam structure.

8. The microchannel for intracellular delivery of functional therapeutic cells according to claim 8, characterized in that: The prepared porous foam structure has a porosity of 10% to 40% and good connectivity.

9. The microchannel for intracellular delivery of functional therapeutic cells according to claim 1, characterized in that: The microchannel, adapter and syringe are assembled, and the sample solution in the syringe is pushed by a propulsion pump to complete the delivery process through the microchannel; the sample solution passing through is a mixture of cells and exogenous functional macromolecules, including cells, cell culture medium and exogenous functional macromolecules.

10. The microchannel for intracellular delivery of functional therapeutic cells according to claim 10, characterized in that: Human T lymphocyte leukemia cells (Jurkat) were selected as a functional therapeutic cell model, and 10 kDa FITC-dextran was used as a model molecule for delivery. The efficiency of delivering 10 kDa FITC-dextran to Jurkat cells was as high as 91.2%, the cell survival rate was as high as 97.2%, and the net acquisition rate was as high as 88.6%.

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