Intracellular delivery device and method for delivering substance into cell

Abstract

The invention discloses an intracellular delivery device and a method for delivering substances into cells, the intracellular delivery device comprises an intermediate ring, the intermediate ring is provided with a centrifugal channel, the centrifugal channel is provided with a first end and a second end which are opposite, and the centrifugal channel is configured to guide cells in a liquid culture medium to move towards the first end in a centrifugal environment; the vessel base is used for closing the first end of the centrifugal channel; the nanoneedle chip is arranged at the first end of the centrifugal channel, and the nanoneedle chip is provided with a tip part for puncturing cells; and the top cover is used for sealing the second end of the centrifugal channel. The intracellular delivery device and the method for delivering the substances into the cells can be suitable for delivery of various cells, and the cell survival rate and the delivery efficiency are relatively high.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an intracellular delivery device and a method for delivering substances into cells. Background Art

[0002] Intracellular molecule delivery technology refers to the technology of delivering various biological molecules, such as nucleic acids, proteins, peptides, small molecule drugs, etc., into cells to achieve specific biological functions or treat diseases.

[0003] The current mainstream technologies include liposome method, electroporation method and viral vector method. Liposome method is the most commonly used method for intracellular delivery, but its delivery efficiency on immune cells and primary cells is low. For electroporation method, its efficiency is higher on difficult-to-transfect cells, but its high electric field intensity leads to high cell mortality rate (about 10-50%). For viral vector method, the delivery efficiency is higher, but the random integration of the viral genome may cause mutations and have a carcinogenic risk. In addition, its cost is high and the production cycle is relatively long (such as 6-8 weeks for lentivirus production).

[0004] A technical solution provides a device for delivering substances, which includes a nanoneedle array. The method achieves substance delivery by enabling the nanoneedle array to move and puncture cells. However, this method requires placing cells in a culture medium on a substrate, the culture medium containing the substance to be delivered; placing a device for delivering substances into the cells on the liquid surface of the culture medium to form a sandwich structure, the device including a substrate and a nanoneedle array attached to the surface of the substrate and spaced apart from each other, the nanoneedle array being formed of diamond; the tips of the nanoneedle array pointing toward the cells; and centrifuging the sandwich structure, so that the centrifugal force causes the tips of the nanoneedle array to penetrate the cells. This nanoneedle array can be used to achieve intracellular delivery, but existing devices and methods are unable to fix the nanoneedle array. Tweezers or other clamping mechanisms are required to place or remove the nanoneedle array, which not only makes operation difficult and prone to contamination, but also causes cells to puncture the nanoneedle array, and removal of the nanoneedle array also makes the cell collection method relatively uncontrollable. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides an intracellular delivery device and a method for delivering a substance into a cell, which has relatively high cell survival rate and delivery efficiency and is relatively simple to operate.

[0006] An intracellular delivery device according to a first embodiment of the present invention is suitable for delivering cells in a suspended state, and the intracellular delivery device comprises:

[0007] a middle ring having a centrifugal channel having opposite first and second ends, wherein the centrifugal channel is configured to guide cells in a liquid culture medium to move toward the first end or the second end under a centrifugal environment;

[0008] a dish base, used for closing the first end of the centrifugal channel;

[0009] a nanoneedle chip, disposed at the first end of the centrifugal channel, the nanoneedle chip having a tip for puncturing cells;

[0010] A top cover is used to close the second end of the centrifugal channel.

[0011] The intracellular delivery device according to the embodiment of the present invention has at least the following beneficial effects:

[0012] The nanoneedle chip is located at the first end of the centrifugal channel and is relatively fixed in a centrifugal environment. The cells suspended in the liquid culture medium move under the action of centrifugal force and are punctured by the tip of the nanoneedle chip to achieve substance delivery. The delivery efficiency is high, the cell survival rate is high, and the cost is relatively low. Since the puncture is achieved by the cells moving in the centrifugal channel, the cells do not need to be fixed, making it suitable for suspended cells and adherent cells; since the nanoneedle chip is located at the first end of the centrifugal channel and cannot move, the cells are detached from the nanoneedle chip by moving them toward the second end in a centrifugal environment, thereby facilitating cell collection; since this intracellular delivery device has good sealing performance, the centrifugal delivery and cell collection steps can be repeated to improve delivery efficiency.

[0013] According to some embodiments of the present invention, the intermediate ring is detachably connected to the dish base, and the nanoneedle chip is sandwiched between the intermediate ring and the dish base.

[0014] According to some embodiments of the present invention, the dish base has a cavity, and the intermediate ring is arranged in the cavity, wherein the inner wall of the dish base has a first threaded portion, and the outer peripheral wall of the intermediate ring has a second threaded portion, and the first threaded portion cooperates with the second threaded portion to connect the intermediate ring to the dish base.

[0015] According to some embodiments of the present invention, a positioning groove is provided on a side of the dish base facing the middle ring, and the nanoneedle chip is disposed in the positioning groove.

[0016] According to some embodiments of the present invention, the outer side wall of the dish base has a third threaded portion, and the top cover has a fourth threaded portion, and the third threaded portion and the fourth threaded portion cooperate to connect the top cover to the dish base.

[0017] According to some embodiments of the present invention, a surface of the top cover facing the middle ring has a groove, and the groove is coaxially arranged with the centrifugal channel and is in communication with each other.

[0018] According to some embodiments of the present invention, it further includes at least one group of elastically deformable sealing rings; one group of sealing rings is a first sealing ring, and the end of the intermediate ring facing the dish base has an injection molding groove surrounding the centrifugal channel, and the first sealing ring is injection molded in the injection molding groove, and the first sealing ring can abut against the dish base along the height direction.

[0019] According to some embodiments of the present invention, at least one group of elastically deformable sealing rings is further included; one group of sealing rings is a second sealing ring, and the top cover has a protruding annular boss on a side facing the intermediate ring, and at least a portion of the second sealing ring is formed on the outer ring surface of the annular boss, and the annular boss and the second sealing ring are both in contact with the intermediate ring in the height direction.

[0020] According to some embodiments of the present invention, the material of the nanoneedle chip is one of silicon, diamond, carbon, gallium nitride, silicon carbide, aluminum oxide, gold, and nickel.

[0021] According to some embodiments of the present invention, the dish base material is polypropylene, polystyrene or ABS resin.

[0022] According to some embodiments of the present invention, the intermediate ring is made of silicone.

[0023] According to a second aspect of the present invention, a method for delivering a substance into a cell is applicable to an intracellular delivery device, and the method comprises:

[0024] placing a liquid culture medium containing cells and a transmitter into a centrifugal channel of the intracellular delivery device;

[0025] placing the intracellular delivery device in a centrifugal environment in a first preset posture for a first preset time period, so that the nanoneedle chip punctures the cells;

[0026] The intracellular delivery device is placed in a centrifugal environment in a second preset posture for a second preset time period, wherein the first preset posture and the second preset posture are in opposite directions.

[0027] According to some embodiments of the present invention, after the step of placing the intracellular delivery device in a centrifugal environment for a second predetermined period of time, the method further comprises:

[0028] Perform the following steps at least once:

[0029] The intracellular delivery device is placed in a centrifugal environment in a first preset posture for a third preset time period, and the intracellular delivery device is placed in a centrifugal environment in a second preset posture for a fourth preset time period.

[0030] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0032] Figure 1 Schematic diagram of the structure of the intracellular delivery device according to an embodiment of the present invention;

[0033] Figure 2 This is an exploded schematic diagram of an intracellular delivery device according to an embodiment of the present invention;

[0034] Figure 3 is a top view of an intracellular delivery device according to an embodiment of the present invention;

[0035] Figure 4 for Figure 3 AA section view;

[0036] Figure 5 These are bright field and fluorescence images of some cells in Example 1 of the method for delivering a substance into cells of the present invention.

[0037] Reference numerals:

[0038] 100, middle ring; 100a, centrifugal channel; 100b, second threaded portion; 100c, injection groove; 110, inner ring body; 120, outer ring body; 130, connecting piece;

[0039] 200, dish base; 200a, first threaded portion; 200b, positioning groove; 200c, third threaded portion;

[0040] 300, nanoneedle chip; 310, substrate; 320, needle;

[0041] 400, top cover; 400a, fourth threaded portion; 400b, groove; 410, annular boss;

[0042] 500, first sealing ring;

[0043] 600, second sealing ring; 610, first ring portion; 620, second ring portion. DETAILED DESCRIPTION

[0044] The following embodiments of the technical solution of the present application will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application and are therefore only examples and are not intended to limit the scope of protection of the present application.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned figure descriptions are intended to cover non-exclusive inclusions.

[0046] In the description of the embodiments of this application, the technical terms "first," "second," "third," etc. are used only to distinguish different objects and should not be understood to indicate or imply relative importance or implicitly specify the quantity, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, the meaning of "plurality" is more than two, unless otherwise specifically defined.

[0047] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0048] In the description of the embodiments of this application, the term "and / or" is simply a description of the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in this document generally indicates that the associated objects are in an "or" relationship.

[0049] In the description of the embodiments of the present application, the orientations or positional relationships indicated by technical terms such as "top", "bottom", "upper", and "lower" are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed, operated or used in a specific orientation. Therefore, they cannot be understood as limitations on the embodiments of the present application.

[0050] In the description of the embodiments of the present application, unless otherwise expressly specified or limited, technical terms such as "installed," "connected," "connect," and "fixed" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integration; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and they may refer to internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the embodiments of the present application based on specific circumstances.

[0051] In the description of the embodiments of the present application, unless otherwise clearly specified and limited, the technical term "contact" should be understood in a broad sense, and can be direct contact, contact through an intermediate medium layer, contact with essentially no interaction force between the two contacting parties, or contact with interaction force between the two contacting parties.

[0052] A technical solution provides a device for delivering substances, which includes a nanoneedle array. The nanoneedle array is activated and punctures cells to achieve substance delivery. However, this method requires placing cells in a culture medium on a substrate, the culture medium containing the substance to be delivered; placing a device for delivering substances into the cells on the surface of the culture medium to form a sandwich structure. The device includes a substrate and a nanoneedle array attached to the surface of the substrate and spaced apart from each other, the nanoneedle array being formed of diamond; the tips of the nanoneedle array are directed toward the cells; and the sandwich structure is centrifuged, where the centrifugal force causes the tips of the nanoneedle array to penetrate the cells. This nanoneedle array can be used to achieve intracellular delivery, but existing devices and methods are unable to fix the nanoneedle array. Tweezers or other clamping mechanisms are required to place or remove the nanoneedle array, which not only makes operation difficult and prone to contamination, but also causes cells to puncture the nanoneedle array, and removal of the nanoneedle array also makes the cell collection method relatively uncontrollable. Furthermore, during centrifugation, the device is open at one end, which is prone to leakage. In addition, due to the limitations of the nanoneedle array preparation process, it is difficult to adapt to the shape of existing culture dishes, resulting in the inability to deliver cells in areas not covered by the nanoneedle array, which will greatly reduce the overall delivery efficiency.

[0053] Furthermore, three experiments were carried out according to the above technical solution, and the results of delivery efficiency and cell survival rate are shown in Table 1.

[0054] Table 1

[0055]

[0056] For this, please refer to Figure 1-Figure 4 An embodiment of the present application provides an intracellular delivery device, including an intermediate ring 100 , a dish base 200 , a top cover 400 and a nanoneedle chip 300 .

[0057] Please refer to Figure 2 and Figure 4 , the middle ring 100 has a centrifugal channel 100a, and the centrifugal channel 100a is used to accommodate a liquid culture medium in which cells are suspended. The centrifugal channel 100a has a first end and a second end relative to each other, and the centrifugal channel 100a is configured to guide the cells in the liquid culture medium to move toward the first end or the second end under a centrifugal environment. That is to say, the intracellular delivery device is placed in a centrifugal environment such as a centrifuge, and the cells in the liquid culture medium can move toward the first end or toward the second end. It should be noted that the specific movement of the cells toward the first end or toward the second end can be achieved by adjusting the posture of the intracellular delivery device in the centrifuge.

[0058] The dish base 200 is used to seal the first end of the centrifugal channel 100a. The top cover 400 is used to seal the second end of the centrifugal channel 100a. It will be appreciated that the dish base 200 and the top cover 400 cooperate to seal both ends of the centrifugal channel 100a, thereby preventing the liquid culture medium in the centrifugal channel 100a from leaking out during centrifugation. When the intracellular delivery device is placed in a centrifuge, either the dish base 200 or the top cover 400 can be positioned downward.

[0059] Nanoneedle chip 300 is located at the first end of centrifugal channel 100a and has a sharp tip for puncturing cells. Under centrifugal conditions, cells can migrate toward nanoneedle chip 300 and be punctured by the tip, enabling substance delivery. Cells can also migrate away from nanoneedle chip 300, allowing them to escape from the chip and be collected.

[0060] In the above embodiment, the nanoneedle chip is located at the first end of the centrifugal channel 100a and is relatively fixed in the centrifugal environment. Cells suspended in the liquid culture medium move under the action of centrifugal force and are punctured by the tip of the nanoneedle chip 300, thereby achieving substance delivery. This achieves high delivery efficiency, high cell survival rate, and relatively low cost. Because the nanoneedle chip is located at the first end of the centrifugal channel and cannot move, the cells are separated from the nanoneedle chip 300 by moving them toward the second end in the centrifugal environment, thereby facilitating cell collection. In addition, there is no need to leave a gap between the nanoneedle chip and the container containing the culture medium for tools to enter. This increases the coverage area of ​​the nanoneedle chip within the centrifugal channel and improves overall delivery efficiency.

[0061] In some embodiments, the intermediate ring 100 is detachably connected to the dish base 200, and the nanoneedle chip 300 is sandwiched between the intermediate ring 100 and the dish base 200. In this way, the intermediate ring 100 can be removed from the dish base 200 and the nanoneedle chip 300 can be removed, making it easier to clean the nanoneedle chip 300 and facilitate subsequent reuse.

[0062] The nanoneedle chip 300 comprises a rectangular substrate 310 and needles 320. The substrate 310 has a circular area at the center of one side of the substrate 310. Multiple needles 320 are connected to the substrate 310 and arranged within the circular area. Along the extension of the centrifugal channel 100a, the projection of the centrifugal channel 100a onto a reference plane overlaps with the projection of the circular area onto the reference plane. This reference plane is a plane perpendicular to the extension of the centrifugal channel 100a.

[0063] The dish base 200 and the intermediate ring 100 may be detachably connected in any manner.

[0064] In some embodiments, the dish base 200 has a cavity, and the intermediate ring 100 is disposed within the cavity. The inner wall of the dish base 200 has a first threaded portion 200a, and the outer wall of the intermediate ring 100 has a second threaded portion 100b. The first threaded portion 200a and the second threaded portion 100b cooperate to connect the intermediate ring 100 to the dish base 200. This allows the dish base 200 and the intermediate ring 100 to be detachable, while also allowing pressure to be applied to the nanoneedle chip 300 during the threaded engagement process to secure the nanoneedle chip 300.

[0065] Exemplarily, the intermediate ring 100 includes an inner ring body 110, an outer ring body 120, and a connecting piece 130 connecting the inner ring body 110 and the outer ring body 120, wherein the inner ring body 110 and the outer ring body 120 are coaxially arranged, the centrifugal channel 100a is located in the inner ring body 110, and the outer peripheral surface of the outer ring body 120 has a second threaded portion 100b.

[0066] In some embodiments, a positioning groove 200b is defined on the surface of the dish base 200 facing the intermediate ring 100, and the nanoneedle chip 300 is positioned within the positioning groove 200b. The shape of the positioning groove 200b is adapted to the base plate portion 310 of the nanoneedle chip 300. The positioning groove 200b allows for quick positioning of the nanoneedle chip 300 relative to the dish base 200, facilitating installation. It also serves to limit the position of the nanoneedle chip 300, preventing displacement when the intermediate ring 100 and dish base 200 are screwed together.

[0067] In some embodiments, the outer wall of the dish base 200 has a third threaded portion 200c, and the top cover 400 has a fourth threaded portion 400a. The third threaded portion 200c and the fourth threaded portion 400a cooperate to connect the top cover 400 to the dish base 200. This not only achieves a detachable connection between the dish base 200 and the top cover 400, but also compresses the intermediate ring 100 to improve the reliability of the sealing of the centrifugal channel 100a.

[0068] In some embodiments, a surface of the top cover 400 facing the middle ring 100 has a groove 400 b , and the groove 400 b is coaxially arranged and communicated with the centrifugal channel 100 a .

[0069] When cells need to be collected, the cells in the liquid culture medium can be moved toward the second end, that is, toward the groove 400b of the top cover 400 by inversion, thereby achieving cell separation and collection. It should be noted that since it is necessary to work in a centrifugal environment, the groove 400b is a circular groove.

[0070] In some embodiments, the dish base 200 is made of polypropylene, polystyrene, or ABS resin.

[0071] In some embodiments, the middle ring 100 is made of silicone.

[0072] In some embodiments, the top cover 400 is made of transparent polycarbonate and is used for pressure balance during centrifugation.

[0073] To enhance sealing, in some embodiments, the intracellular delivery device includes at least one set of elastically deformable sealing rings. One set of sealing rings is a first sealing ring 500. The end of the intermediate ring 100 facing the dish base 200 includes an injection molding groove 100c surrounding the centrifugal channel 100a. The first sealing ring 500 is injection molded within the injection molding groove 100c. The first sealing ring 500 is capable of abutting the dish base 200 along the height direction to seal the first end of the centrifugal channel 100a.

[0074] In some embodiments, one of the sealing rings is a second sealing ring 600, and the top cover 400 has a protruding annular boss 410 on the side facing the intermediate ring 100, and the middle part of the annular boss 410 is a groove 400b. At least a portion of the second sealing ring 600 is formed on the outer annular surface of the annular boss 410. The annular boss 410 and the second sealing ring 600 are both in contact with the intermediate ring 100 in the height direction, thereby closing the second end of the centrifugal channel 100a.

[0075] Specifically, in some embodiments, the second sealing ring 600 includes a first ring portion 610, a second ring portion 620, and a connecting portion. The first ring portion 610 is located on the outer annular surface of the annular boss 410, the second ring portion 620 is located on the side of the top cover 400 facing away from the intermediate ring 100, and the connecting portion is used to connect the first ring portion 610 and the second ring portion 620. An annular groove for mounting the second ring portion 620 is formed on the side of the top cover 400 facing away from the intermediate ring 100, and a through hole is formed at the bottom of the annular groove for the connection portion.

[0076] The present invention also provides a method for delivering a substance into a cell, which is suitable for Figure 1-Figure 4 The intracellular delivery device of the present invention comprises:

[0077] S1. placing a liquid culture medium containing cells and transmitter into a centrifugal channel of an intracellular delivery device;

[0078] S2. Placing the intracellular delivery device in a centrifugal environment in a first preset posture for a first preset time period.

[0079] The specific process of step S1 is as follows: the nanoneedle chip is placed on the dish base, and the intermediate ring is connected to the dish base, so that the nanoneedle chip is at the first end of the centrifugal channel, and the dish base closes the first end. Liquid culture medium containing cells is placed in the centrifugal channel. The top cover is connected to the dish base to close the second end of the centrifugal channel.

[0080] In step S2, the first preset duration is 3-10 minutes. In some specific embodiments, the first preset duration is 5 minutes.

[0081] The centrifugal environment specifically includes a centrifuge speed of 300-3000 rpm. In some embodiments, the centrifuge is a Sorvall ST 16R. The speed is adjusted according to different cells.

[0082] In some embodiments, when the cells are adherent cells, before step S1, the following steps are included:

[0083] Aspirate the culture medium from the cell culture plate, rinse the cell surface 1-2 times with PBS (phosphate-buffered saline), and digest the cells with 0.25% trypsin-EDTA (enough to cover the culture surface, e.g., 1 mL for a T25 flask). Incubate at 37°C for 1-3 minutes (stop once cells shrink and become rounded under a microscope). Terminate trypsinization by adding serum-containing culture medium and pipette to form a single-cell suspension. Collect the cells by centrifugation and resuspend in the desired liquid culture medium.

[0084] In some embodiments, after step S2, the method further includes:

[0085] S3. Place the intracellular delivery device in a centrifugal environment in a second preset posture for a second preset time period, wherein the first preset posture and the second preset posture are in opposite directions. That is, after step S2, turn the intracellular delivery device upside down and then place it in the centrifugal environment again.

[0086] In step S3, the second preset duration is 3-10 minutes. In some specific embodiments, the first preset duration is 5 minutes. The relevant parameters in step S3 can be selected to be the same as those in step 2.

[0087] After step S3, the cells were washed three times with PBS (phosphate buffered saline) and cultured at 37° C. for 24 hours to detect the expression of the delivered gene protein.

[0088] The cells were washed three times with PBS to remove residual culture medium, serum, and other impurities on the cell surface to prevent them from interfering with subsequent experiments. 37°C is the normal physiological temperature of human cells, at which they maintain good metabolic and physiological activity. The 24-hour incubation period allows the cells ample time to carry out various physiological activities, including protein expression of relevant genes.

[0089] In some embodiments, after step S3, the following steps are included:

[0090] Perform the following steps at least once:

[0091] S4. Placing the intracellular delivery device in a centrifugal environment in a first preset posture for a third preset time period, and placing the intracellular delivery device in a centrifugal environment in a second preset posture for a fourth preset time period. The third preset time period may be the same as or different from the first preset time period, and the fourth preset time period may be the same as or different from the second preset time period.

[0092] It can be understood that in the above steps, placing the intracellular delivery device in the centrifugal environment again in the first preset posture can enable the nanoneedle chip to pierce the cells multiple times to achieve delivery, thereby further improving the cell delivery efficiency and also increasing the number of cells delivered by the intracellular delivery device in a single time.

[0093] Example 1: GFP plasmid was delivered into a variety of cells, including A549 cells, HEK293 cells, SH-SY5Y cells, Jurkat cells, U937 cells, and HL-60 cells.

[0094] Objective: To verify the gene delivery efficiency and cell survival rate of the present invention in different types of cells.

[0095] Implementation steps and parameters

[0096] Materials preparation:

[0097] Cells: A549 cells, HEK293 cells, SH-SY5Y cells (adherent culture), Jurkat cells, U937 cells, HL-60 cells (suspension culture).

[0098] Operation density: 4×10 6 cells / mL.

[0099] Delivery molecule: GFP plasmid (5 μg / mL, dissolved in PBS).

[0100] Parameters of the intracellular delivery device: nanoneedles (density 4×106 / cm 2 ).

[0101] Process flow:

[0102] Step 1: Inject a cell and plasmid mixture (100 μL of cell suspension, 100 μL of plasmid mixture) into the central cavity of the delivery dish and install a silicone sealing ring.

[0103] Step 2: Centrifuge at 300 rpm (RCF = 50 x g) for 5 minutes (25°C). 300 rpm indicates a centrifuge speed of 300 revolutions per minute, and RCF = 50 x g indicates a relative centrifugal force of 50 times the acceleration of gravity. Step 3: Invert the dish and centrifuge again (using the same parameters) to recover the cells.

[0104] Step 4: Wash the cells three times with PBS and culture them at 37°C for 24 hours. Count the cells under a fluorescence microscope or detect GFP expression by flow cytometry.

[0105] In the above Example 1, the results of the delivery efficiency and cell survival rate are shown in Table 2. According to the comparison results of Table 2 and Table 1, it can be seen that the structure of the present application can improve the delivery efficiency and cell survival rate.

[0106] Table 2

[0107]

[0108] Figure 5 Some of the results are shown in Table 2.

[0109] In Table 2, the delivery efficiency is obtained by delivering GFP plasmid to cells and observing them under a fluorescence microscope 48 hours after transfection to obtain bright field and fluorescence images. The delivery efficiency can be obtained by displaying the percentage of GFP-positive cells by flow cytometry. For easier understanding, please refer to Figure 5 , Figure 5 Bright field and fluorescence images of some cells are shown in Figure .

[0110] Example 2: Delivery of CAR plasmids to T cells

[0111] Objective: To verify the gene delivery efficiency and cell survival rate of CAR-T cells under different cell operation densities, different centrifugation times, and different centrifugation conditions of the present invention.

[0112] Implementation steps and parameters

[0113] Materials preparation:

[0114] Cells: Jurkat T cells (suspension culture, operating density 4×10 6 cells / mL).

[0115] Delivery molecule: anti-CD19 CAR plasmid (5 μg / mL, dissolved in PBS).

[0116] Parameters of the intracellular delivery device: nanoneedles (density 4×106 / cm 2 ).

[0117] Process flow:

[0118] Step 1: Inject the cell and plasmid mixture (100 μL of cell suspension, 100 μL of plasmid mixture) into the center cavity of the delivery dish and install a silicone sealing ring.

[0119] Step 2: Set the centrifuge to 300 rpm (RCF = 50×g) - 500 rpm (RCF = 120×g) and centrifuge for 3 minutes - 5 minutes (25°C).

[0120] Step 3: Invert the delivery dish and centrifuge again (with the same parameters) to recover the cells.

[0121] Step 4: Wash the cells three times with PBS and culture them at 37°C for 24 hours to detect CAR expression.

[0122] Experimental results:

[0123]

[0124]

[0125] Conclusion: The present invention can select different operating densities, centrifugation times, and centrifugation speeds for different cells, thereby improving cell delivery efficiency.

[0126] Example 3: Delivery of CAR plasmids to T cells

[0127] Objective: To verify the gene delivery efficiency, survival rate and cost of the present invention in CAR-T cells.

[0128] Implementation steps and parameters

[0129] Materials preparation:

[0130] Cells: Jurkat T cells (suspension culture, operating density 4×10 6 cells / mL).

[0131] Delivery molecule: anti-CD19 CAR plasmid (5 μg / mL, dissolved in PBS).

[0132] Parameters of the intracellular delivery device: nanoneedles (density 4×106 / cm 2 ).

[0133] Process flow:

[0134] Step 1: Inject the cell and plasmid mixture (100 μL of cell suspension, 100 μL of plasmid mixture) into the center cavity of the delivery dish and install a silicone sealing ring.

[0135] Step 2: Set the centrifuge at 300 rpm (RCF = 50×g) and centrifuge for 5 minutes (25° C.).

[0136] Step 3: Invert the delivery dish and centrifuge again (with the same parameters) to recover the cells.

[0137] Step 4: Wash the cells three times with PBS and culture them at 37°C for 24 hours to detect CAR expression.

[0138] Experimental results:

[0139] index Example 3 Lentiviral vector method Delivery efficiency 85％±3％ 70％±5％ Cell viability 92％±2％ 60％±8％ production costs 50RMB / time 1,000 RMB / time Preparation cycle 30 minutes 5 days

[0140] Conclusion: The present invention can improve delivery efficiency and cell survival in CAR-T preparation, and can significantly reduce costs. Comparative Example 1: Delivery of CAR Plasmids to T Cells Objective: To verify the gene delivery efficiency of CAR-T cell therapy when gene delivery is performed under the conditions of double centrifugation in the present invention.

[0141] Implementation steps and parameters

[0142] Materials preparation:

[0143] Cells: Jurkat T cells (suspension culture, operating density 8×10 6 cells / mL).

[0144] Delivery molecule: anti-CD19 CAR plasmid (5 μg / mL, dissolved in PBS).

[0145] Parameters of the intracellular delivery device: nanoneedles (density 4×10 6 / cm 2 ).

[0146] Process flow:

[0147] Step 1: Inject the cell and plasmid mixture (100 μL of cell suspension, 100 μL of plasmid mixture) into the center cavity of the delivery dish and install a silicone sealing ring.

[0148] Step 2: Set the centrifuge at 300 rpm (RCF = 50×g) and centrifuge for 5 minutes (25° C.).

[0149] Step 3: Invert the delivery dish and centrifuge again (with the same parameters) to recover the cells.

[0150] Step 4: Invert the delivery dish and centrifuge three times (same parameters) for secondary delivery.

[0151] Step 5: Invert the delivery dish and centrifuge four times (with the same parameters) to recover the cells.

[0152] Step 6: Wash the cells three times with PBS and culture them at 37°C for 24 hours to detect CAR expression.

[0153] Experimental results:

[0154] index Example 3 Comparative Example 1 Delivery efficiency 85％±3％ 90±3％ Cell viability 90％±2％ 85％±8％ Amount of cells delivered per time <![CDATA[4×10 5 ]]> <![CDATA[8×10 5 ]]> Preparation cycle 30 minutes 1 hour

[0155] Conclusion: When gene delivery was performed under the conditions of two centrifugations, the CAR-T preparation efficiency was increased by 5%, the survival rate was reduced by 5%, and the amount of cells delivered in a single delivery was twice as much as before.

[0156] Comparative Example 2: Delivery of CAR plasmid to T cells Purpose: To verify the gene delivery efficiency in CAR-T cell therapy when gene delivery is performed under the three centrifugation conditions of the present invention.

[0157] Implementation steps and parameters

[0158] Materials preparation:

[0159] Cells: Jurkat T cells (suspension culture, operating density 1.2×10 7 cells / mL).

[0160] Delivery molecule: anti-CD19 CAR plasmid (5 μg / mL, dissolved in PBS).

[0161] Parameters of the intracellular delivery device: nanoneedles (density 4×106 / cm 2 ).

[0162] Process flow:

[0163] Step 1: Inject the cell and plasmid mixture (100 μL of cell suspension, 100 μL of plasmid mixture) into the center cavity of the delivery dish and install a silicone sealing ring.

[0164] Step 2: Set the centrifuge at 300 rpm (RCF = 50×g) and centrifuge for 5 minutes (25° C.).

[0165] Step 3: Invert the delivery dish and centrifuge again (with the same parameters) to recover the cells.

[0166] Step 4: Invert the delivery dish and centrifuge three times (same parameters) for secondary delivery.

[0167] Step 5: Invert the delivery dish and centrifuge four times (with the same parameters) to recover the cells.

[0168] Step 6: Invert the delivery dish and centrifuge five times (same parameters) to perform three deliveries.

[0169] Step 7: Invert the delivery dish and centrifuge six times (with the same parameters) to recover the cells.

[0170] Step 8: Wash the cells three times with PBS and culture them at 37°C for 24 hours to detect CAR expression.

[0171] Experimental results:

[0172] index Example 3 Comparative Example 2 Delivery efficiency 85％±3％ 95±3％ Cell viability 90％±2％ 80％±8％ Amount of cells delivered per time <![CDATA[4×10 5 ]]> <![CDATA[1.2×10 6 ]]> Preparation cycle 30 minutes 1.5 hours

[0173] Conclusion: When gene delivery was performed under three centrifugation conditions, the CAR-T preparation efficiency was increased by 10%, the survival rate was reduced by 10%, and the amount of cells delivered in a single delivery was three times the original amount.

[0174] Example 4: Antibody delivery to NK cells

[0175] Objective: To verify the broad spectrum molecular delivery capability (large molecular weight protein).

[0176] Implementation steps and parameters

[0177] Material:

[0178] Cells: NK-92 cells (suspension, operating density 5×10 5 cells / mL).

[0179] Delivery molecule: Fluorescently labeled anti-PD-1 antibody (10 μg / mL).

[0180] Parameters of the intracellular delivery device: nanoneedles (density 5×10 5 / cm 2 ).

[0181] Process parameters:

[0182] Centrifugal force: 500 rpm (RCF = 120 × g), centrifugation time: 10 minutes.

[0183] Temperature control: Operate at 4°C throughout the process to reduce antibody denaturation.

[0184] Experimental results:

[0185] index Example 4 Electroporation Antibody intracellular penetration 78％±4％ 30％±5％ Cell death rate 3％±1％ 40％±6％ Device Dependency Ordinary centrifuge Dedicated electroporator

[0186] Conclusion: Antibody delivery efficiency was increased by 160%, and mortality was reduced to 7.5% of electroporation.

[0187] Example 5: Small molecule drug delivery to B cells (low-cost validation)

[0188] Objective: To verify the applicability and reproducibility of small molecule delivery.

[0189] Implementation steps and parameters

[0190] Material:

[0191] Cells: Raji B cells (suspension, operating density 2×10 6 cells / mL).

[0192] Delivery molecule: Doxorubicin (1 μM).

[0193] Alternative solution: nanoneedles (density 2×10 6 / cm 2 ).

[0194] Process parameters:

[0195] Centrifugal force: 1000 rpm (RCF = 200 × g), time: 2 minutes.

[0196] Reusability: The same delivery dish is used 50 times in a row to test its performance.

[0197] index First use 50th use Intracellular drug concentration (nM) 950±50 920±60 Cell viability 95％±2％ 93％±3％ Nanoneedle wear rate 0％ <5％

[0198] Conclusion: The performance of the delivery dish decreased by <3% after 50 reuses, and the cost per use was reduced to $1.

[0199] Solution: Replacement of dish material (polypropylene vs. polystyrene)

[0200] parameter polystyrene Polypropylene Centrifugal resistance 3000rpm without deformation 2000rpm slight deformation Chemical compatibility Resistant to organic solvents Partial solvent dissolution

[0201] Through the above embodiments and alternative solutions, the core advantages of the present invention are:

[0202] Broad-spectrum applicability: Supports transmembrane delivery of molecules ranging from 1 kDa small molecules (doxorubicin) to 100 kDa large molecules (antibodies). High efficiency and low damage: CAR plasmid delivery efficiency exceeds 85%, and cell survival rate exceeds 90%, significantly outperforming viral and electroporation methods. Cost-effective: Single-use cost starts at 8 RMB and can be reused over 50 times.

[0203] Flexible process: The nanoneedle material and dish body material (PS / PP / ABS) can be replaced to adapt to different scene requirements.

[0204] In addition, the present application further provides experimental data on cell delivery to demonstrate that the present device is applicable to the delivery of different types of cells.

[0205]

[0206]

[0207] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application, and they should all be included in the scope of the specification of the present application. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions that fall within the scope of protection.

Claims

1. An intracellular delivery device, characterized in that Suitable for delivery of cells in suspension, the intracellular delivery device comprises: a middle ring having a centrifugal channel having opposite first and second ends, wherein the centrifugal channel is configured to guide cells in a liquid culture medium to move toward the first end or the second end under a centrifugal environment; a dish base, used for closing the first end of the centrifugal channel; a nanoneedle chip, disposed at the first end of the centrifugal channel, the nanoneedle chip having a tip for puncturing cells; A top cover is used to close the second end of the centrifugal channel.

2. The intracellular delivery device according to claim 1, wherein The middle ring is detachably connected to the dish base, and the nano needle chip is clamped between the middle ring and the dish base.

3. The intracellular delivery device according to claim 2, characterized in that The dish base has a cavity, and the intermediate ring is arranged in the cavity, wherein the inner wall of the dish base has a first threaded portion, and the outer wall of the intermediate ring has a second threaded portion, and the first threaded portion cooperates with the second threaded portion to connect the intermediate ring to the dish base.

4. The intracellular delivery device according to claim 2, characterized in that A positioning groove is provided on a side of the dish base facing the middle ring, and the nano needle chip is arranged in the positioning groove.

5. The intracellular delivery device according to claim 1, wherein The outer wall of the dish base has a third threaded portion, and the top cover has a fourth threaded portion. The third threaded portion and the fourth threaded portion cooperate to connect the top cover to the dish base.

6. The intracellular delivery device according to claim 1, characterized in that A surface of the top cover facing the middle ring is provided with a groove, and the groove and the centrifugal channel are coaxially arranged and communicated with each other.

7. The intracellular delivery device according to claim 1, characterized in that Also includes at least one set of elastically deformable sealing rings; One of the sealing rings is a first sealing ring, and the end of the intermediate ring facing the dish base has an injection groove surrounding the centrifugal channel, and the first sealing ring is injection-molded in the injection groove. The first sealing ring can abut against the dish base along the height direction; and / or, One group of the sealing rings is the second sealing ring, and the top cover has a protruding annular boss on one side facing the intermediate ring. At least part of the second sealing ring is formed on the outer ring surface of the annular boss, and the annular boss and the second sealing ring are both in contact with the intermediate ring along the height direction.

8. The intracellular delivery device according to claim 1, wherein The material of the nanoneedle chip is one of silicon, diamond, carbon, gallium nitride, silicon carbide, aluminum oxide, gold, and nickel; and / or, The dish base material is polypropylene, polystyrene or ABS resin; and / or, The material of the intermediate ring is silicone.

9. A method for delivering a substance into a cell, characterized in that: Suitable for the intracellular delivery device according to any one of claims 1 to 8, the method comprising: placing a liquid culture medium containing cells and a transmitter into a centrifugal channel of the intracellular delivery device; placing the intracellular delivery device in a centrifugal environment in a first preset posture for a first preset time period, so that the nanoneedle chip punctures the cells; The intracellular delivery device is placed in a centrifugal environment in a second preset posture for a second preset time period, wherein the first preset posture and the second preset posture are in opposite directions.

10. The method for delivering a substance into a cell according to claim 9, wherein After the step of placing the intracellular delivery device in a centrifugal environment for a second predetermined period of time, the method further comprises: Perform the following steps at least once: The intracellular delivery device is placed in a centrifugal environment in a first preset posture for a third preset time period, and the intracellular delivery device is placed in a centrifugal environment in a second preset posture for a fourth preset time period.

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