Nucleic acid transfection reagent for suspension cells as well as preparation method and application of nucleic acid transfection reagent

Abstract

The invention discloses a nucleic acid transfection reagent for suspension cells as well as a preparation method and application of the nucleic acid transfection reagent. Nucleic acid molecule groups can be delivered to the suspension cells. The transfection reagent is mainly composed of a nucleic acid delivery polymer and a suspension cell specific substance, the structure of the transfection reagent is vesicles or micelles, and nucleic acid is adsorbed on the surface of the reagent through electrostatic interaction. The suspension cell specific substance in the reagent promotes the combination of the transfection reagent and the suspension cell surface characteristic group, improves the binding rate of the transfection reagent and the suspension cell, and realizes efficient nucleic acid delivery. The transfection reagent prepared by the invention overcomes the problem of low nucleic acid delivery efficiency of a transfection reagent in suspension cells in the prior art, and has good biocompatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of nanobiomaterials, and particularly relates to a nucleic acid transfection reagent for suspension cells, a preparation method thereof, and an application thereof. Background Art

[0002] Suspension cells grow independently of a support and grow and reproduce in a suspension state in a culture medium (such as MOLM13, hematopoietic stem cells, NK cells, and T cells, etc.). Compared with adherent cells, continuously flowing suspension cells are more difficult to transfect. First of all, suspension cells have a smaller cell surface area, higher floating and motility, and these characteristics will reduce the contact opportunity and binding probability between suspension cells and transfection vectors, resulting in low transfection efficiency of transfection reagents for suspension cells. Secondly, suspension cells are more difficult to culture and more prone to death compared with adherent cells. Therefore, the method of cell transfection mediated by substances with high cytotoxicity will affect the normal growth and function of cells.

[0003] Cationic liposomes are the most commonly used non-viral nucleic acid delivery vectors at present. When cationic liposomes are applied to cell transfection reagents, the contact opportunity with adherent cells is much higher than that with suspension cells. Therefore, the transfection efficiency of cationic liposomes in suspension cells is low. In particular, liposomes have strong toxicity to suspension cells, resulting in low delivery efficiency of nucleic acids by liposomes and low cell viability. Therefore, it is very difficult for cationic liposomes to be used as nucleic acid delivery vectors for the transfection of suspension cells.

[0004] Based on the above technical problems, the present invention provides a nucleic acid transfection reagent that can be used for suspension cells, which can achieve efficient nucleic acid transfection of suspension cells, and the highest transfection efficiency can reach more than 80%, thus overcoming the difficulty of the currently commercially available transfection reagents in efficiently delivering nucleic acids to suspension cells. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a nucleic acid transfection reagent for suspension cells, a preparation method thereof, and an application thereof, which not only have the characteristics of safety and high efficiency.

[0006] To achieve the above purpose, the first aspect of the present invention provides a nucleic acid transfection reagent for transfection of suspension cells. The nucleic acid transfection reagent is a nanoparticle assembled from a nucleic acid delivery polymer and a suspension cell-specific substance; wherein, the surface of the nucleic acid transfection reagent is positively charged, and can adsorb negatively charged nucleic acids on the surface of the nanoparticle through electrostatic interaction. The suspension cell-specific substance can bind to the surface characteristic groups of suspension cells; the nucleic acid transfection reagent can load nucleic acids to transfect suspension cells and achieve nucleic acid transfection of suspension cells; the positive charge comes from the nucleic acid delivery polymer.

[0007] Preferably, the nucleic acid delivery polymer and the suspension cell-specific substance are assembled by hydrophobic interaction at a volume ratio of 1:1 to 20 to obtain the nanoparticles.

[0008] Preferably, the nanoparticles are any one of nano micelles and vesicles.

[0009] Preferably, the particle size of the nanoparticles is 5 to 1000 nm.

[0010] Preferably, the nanoparticles are prepared by any one of ultrasonic dispersion method, ultrasonic emulsification method, thin film dispersion method, mechanical stirring method and microfluidic method.

[0011] Preferably, the surface of the nanoparticles has nucleic acid binding sites, and the nucleic acid binding sites are derived from the suspension cell-specific substance. The nucleic acid binds to the nucleic acid transfection reagent through the nucleic acid binding sites to achieve the transport of the nucleic acid.

[0012] Preferably, the suspension cell-specific substance includes but is not limited to at least one of dodecyl aldehyde, dopamine hydrochloride, stearic acid N-hydroxysuccinimide ester, 2,5-dioxopyrrolidin-1-yl myristate, palmitic acid hydrazide, palmitic anhydride, dodecyl aldehyde, N,N'-dicyclohexylcarbodiimide, DHSO (3,3'-dithiobis(propanehydrazide)), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-succinimidyl myristate, sodium palmitate, palmitic acid N-hydroxysuccinimide ester, DOTMA lipid, disuccinimidylsuberate, 1-(3-mercapropropanoyl-N-hydroxysuccinimide ester).

[0013] The addition of the suspension cell-specific lipid increases the binding rate between the transfection reagent and the suspension cells. The possible reason is that the suspension cell-specific substance has a stronger affinity with the surface characteristic groups of the suspension cells, thereby promoting the phagocytosis of the nanoparticles by the suspension cells ( Nature Nanotechnology , 2024, 19 : 1409-1417).

[0014] Preferably, the nucleic acid delivery polymer is slightly soluble in water, has lipophilicity, and has nucleic acid binding sites.

[0015] More preferably, the nucleic acid delivery polymer comprises a poly(β-amino ester). The poly(β-amino ester) has acid-responsive properties, which can promote the response of the nucleic acid transfection reagent to pH, facilitate the escape of the lysosome after the transfection reagent-nucleic acid complex is phagocytosed by cells, promote the release of nucleic acid into the cytoplasm, and thus achieve efficient cell transfection, with the highest transfection efficiency reaching over 80%.

[0016] More preferably, the molecular weight of the nucleic acid delivery polymer is 100 - 200,000 Da.

[0017] Preferably, the nucleic acid includes but is not limited to at least one of small molecule messenger nucleic acid, mRNA, reporter gene mRNA, therapeutic gene mRNA, interfering gene plasmid, therapeutic protein nucleic acid plasmid, reporter gene plasmid, dsRNA, antisense nucleic acid, antisense oligonucleotide, microRNA, antisense microRNA, microRNA inhibitor, microRNA activator, and immune-stimulating nucleic acid.

[0018] As another aspect of the present invention, the present invention also provides a method for preparing the above-mentioned nucleic acid transfection reagent for suspension cells, which specifically includes the following steps: S1. Add a suspension cell-specific substance and a poly(β-amino ester) to a solvent and mix to obtain a mixed solution; S2. In an ultrasonic environment, add the mixed solution to pure water and mix evenly, and perform purification treatment to obtain the nucleic acid transfection reagent.

[0019] Preferably, in S1, in the mixed solution, the mass ratio of the suspension cell-specific substance to the poly(β-amino ester) is 1:5 - 20.

[0020] Preferably, in S2, the solvent is any one of ethanol, dichloromethane, chloroform, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide.

[0021] Preferably, in S2, the method for mixing the mixed solution with water includes any one of ultrasonic emulsification method, thin film dispersion method, mechanical stirring method, and microfluidic method.

[0022] Preferably, the purification treatment is ultrafiltration purification treatment.

[0023] As one aspect of the present invention, the present invention also provides a method for transfecting nucleic acid into suspension cells, which includes mixing a cationic lipid, a nucleic acid delivery polymer, and an amphiphilic polymer to form a nucleic acid transfection reagent as a nano-delivery carrier, using the nucleic acid transfection reagent to transport nucleic acid into suspension cells, and the escape of the lysosome after the transfection reagent-nucleic acid complex is phagocytosed by cells, promoting the release of nucleic acid into the cytoplasm, thereby achieving efficient cell transfection.

[0024] Furthermore, after the nucleic acid transfection reagent is co-incubated with the nucleic acid, it is added to the suspension cells, and after mixing evenly, the cells are cultured.

[0025] Specifically, the transfection method includes: the specific steps include: first, mixing and incubating the nucleic acid with sodium acetate for 5-10 minutes to obtain a nucleic acid mixture; adding the nucleic acid mixture to the nucleic acid transfection reagent mixture, mixing evenly, and then co-incubating for 5-10 minutes to obtain a transfection mixture; adding the transfection mixture to the culture medium and mixing thoroughly to form a transfection culture solution; finally, replacing the suspension cell culture medium with the transfection culture solution and culturing for 1-24 hours, thus completing the nucleic acid transfection of the suspension cells.

[0026] The beneficial technical effects obtained by the present invention are as follows: (1) By adopting the technical solution of the present invention, a transfection reagent that can be used for efficient nucleic acid transfection of suspension cells is provided, which can efficiently deliver nucleic acids into difficult-to-transfect suspension cells, with a transfection efficiency of more than 80%, effectively solving the problem of low efficiency of nucleic acid delivery to suspension cells in the prior art; and it does not affect cell function, and the cell survival rate is more than 90%.

[0027] (2) In the composition of the nucleic acid transfection reagent of the present invention, the use of cationic liposomes is avoided, and only suspension cell-specific substances and poly-β-amino esters are selected. The suspension cell-specific substances bind to the characteristic groups on the surface of suspension cells to improve the binding efficiency of the transfection reagent to suspension cells to achieve efficient nucleic acid delivery to suspension cells; while poly-β-amino esters have acid-responsive properties, which are beneficial to the escape of the transfection reagent-nucleic acid complex from lysosomes after being phagocytosed by cells, promoting the release of nucleic acids into the cytoplasm, thereby achieving efficient cell transfection. The nucleic acid transfection reagent provided by the present invention has the characteristics of simple composition and high transfection efficiency.

[0028] (3) The nucleic acid transfection reagent provided by adopting the technical solution of the present invention not only has good biocompatibility, but also can be used for the labeling and tracing of suspension cells.

[0029] (4) The raw materials of the nucleic acid transfection reagent of the present invention are simple and easy to obtain, and are very safe. The whole transfection process is convenient and controllable, and it is suitable for industrial production. Description of the Drawings

[0030] Figure 1 It is a schematic diagram of the simulated structure of the nucleic acid transfection reagent prepared by the present invention.

[0031] Figure 2 It is a transmission electron micrograph of the transfection reagent-1 loaded with nucleic acid prepared in Example 1 of the present invention.

[0032] Figure 3It is a fluorescence and flow cytometry quantification result graph in the cytoplasm after the transfection reagent-1 prepared in Example 1 of the present invention and Lipo6000 were co-incubated with mRNA expressing green fluorescent protein (GFP-mRNA) and then transfected into MOLM13 cells for 12 hours.

[0033] Figure 4 It is a fluorescence result graph 12 hours after the transfection reagent-2 prepared in Example 2 of the present invention was co-incubated with a plasmid expressing red fluorescent protein (mCherry plasmid) and then transfected into hematopoietic stem cells.

[0034] Figure 5 It is a comparative graph of bioluminescence results 24 hours after the transfection reagent-3 prepared in Example 3 of the present invention and Lipo6000 were respectively co-incubated with a plasmid expressing red firefly luciferase (CAR plasmid) and transfected into NK cells.

[0035] Figure 6 It is a fluorescence graph in the cytoplasm 12 hours after the transfection reagents prepared in Examples 4-10 of the present invention were co-incubated with mRNA expressing green fluorescent protein (GFP-mRNA) and then transfected into MOLM13 cells. Detailed implementation mode

[0036] In view of the defects of the prior art, the present invention provides a nucleic acid transfection reagent for suspension cells, which is composed of a nucleic acid delivery polymer and a suspension cell-specific substance, and uses an ultrasonic emulsification method to form a nanocarrier with a micelle structure, and the nucleic acid is adsorbed on its surface. The suspension cell-specific substance binds to the characteristic groups on the surface of the suspension cells to improve the binding efficiency of the transfection reagent to the suspension cells, realizing safe and efficient nucleic acid delivery, and overcoming the problem that the currently commercially available transfection reagents are difficult to target and bind to suspension cells to achieve efficient nucleic acid delivery.

[0037] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.

[0038] One aspect of the embodiments of the present invention provides a transfection reagent for efficient transfection of nucleic acids into cells, especially for suspension cells, which includes: The transfection reagent is mainly composed of a suspension cell-specific substance and a nucleic acid delivery polymer, forming micelle or vesicle-like nanoparticles, and the nucleic acid binds to the transfection reagent through electrostatic interaction. Refer to Figure 1, which is a schematic structural simulation diagram of the nucleic acid transfection reagent provided by the present invention. As can be seen from the figure, the transfection reagent provided by the present invention is mainly composed of nucleic acid delivery polymers and nanometer particles composed of suspension cell-specific substances, and has nucleic acid binding sites on the surface. The nucleic acid binding sites mainly come from the nucleic acid delivery polymers; nucleic acids are bound to the nucleic acid transfection reagent through the nucleic acid binding sites to achieve the transportation of nucleic acids; the suspension cell-specific substances can bind to the surface characteristic groups of suspension cells, thereby realizing the transfection of nucleic acids.

[0039] In some more specific embodiments, the nucleic acid delivery polymers and the suspension cell-specific substances are assembled into the nanometer particles through hydrophobic interaction at a volume ratio of 1:1 to 20.

[0040] In some more specific embodiments, the suspension cell-specific substances include at least one of dodecylaldehyde, dopamine hydrochloride, stearic acid-N-hydroxysuccinimide ester, 2,5-dioxopyrrolidin-1-yl myristate, palmitic acid hydrazide, palmitic anhydride, dodecyl aldehyde, N,N'-dicyclohexylcarbodiimide, DHSO(3,3′-sulfinyldi(propanehydrazide)), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-succinimidyl myristate, 1,4-phthalaldehyde, sodium palmitate, palmitic acid N-hydroxysuccinimide ester, DOTMA lipid, disuccinimidyl suberate, 1-(3-mercapto-1-oxopropoxy)-2,5-pyrrolidinedione (3-mercapropropanoyl-N-hydroxysuccinimide ester).

[0041] Furthermore, the nucleic acid delivery polymer is poly-β-amino ester.

[0042] Furthermore, the nucleic acids include one of small molecule messenger nucleic acids, mRNAs, reporter gene mRNAs, therapeutic gene mRNAs, interfering gene plasmids, therapeutic protein nucleic acid plasmids and reporter gene plasmids, dsRNAs, antisense nucleic acids, antisense oligonucleotides, microRNAs, antisense microRNAs, microRNA inhibitors, microRNA activators and immunostimulatory nucleic acids.

[0043] Furthermore, the transfection reagent is prepared from suspension cell-specific substances and poly-β-amino ester by ultrasonic emulsification method, thin film dispersion method, mechanical stirring method or microfluidic method.

[0044] Furthermore, the molecular weight of the poly-β-amino ester is 100 - 200000 Da.

[0045] In the present invention, the poly-β-amino ester and the suspended cell-specific substance form nano micelles or vesicles, wherein the suspended cell-specific lipid can efficiently target suspended cells; the poly-β-amino ester and the suspended cell-specific substance are acid-responsive substances, and after the transfection reagent-nucleic acid complex is phagocytosed by cells, the nucleic acid escapes from lysosomes through the proton sponge effect, enabling the nucleic acid to enter the cytoplasm faster and further expressing.

[0046] In the present invention, nucleic acids are bound to the surface of the transfection reagent through electrostatic interaction, enabling efficient transfection of suspended cells.

[0047] Another aspect of the embodiments of the present invention also provides a preparation method of the aforementioned transfection reagent, which includes: The method for synthesizing nanoparticles is prepared by ultrasonic emulsification, and its synthesis steps are as follows: 1. Add the suspended cell-specific substance (10 mg / mL) and PBAE (100 mg / mL) to tetrahydrofuran (THF) in proportion to obtain solution A; 2. In an ultrasonic environment, add the above solution to water in multiple small portions for purification treatment to obtain the transfection reagent.

[0048] In some more specific embodiments, the organic solvent includes any one or a combination of two or more of ethanol, tetrahydrofuran, dimethyl sulfoxide, dichloromethane, chloroform, N,N-dimethylformamide, and is not limited thereto.

[0049] In some more specific embodiments, the preparation method includes: in an ultrasonic environment, add solution A to water for mixing, homogenization, ultrasonic treatment, and purification treatment.

[0050] Furthermore, solution A is added to water in small portions multiple times, and the addition time is 0.1 - 20 min; preferably, it is completed within 1 min.

[0051] Furthermore, the purification treatment is ultrafiltration purification treatment.

[0052] In some more specific embodiments, when applied to cell transfection, the nucleic acid transfection reagent is mixed with nucleic acids and then used for cell transfection.

[0053] In some embodiments, the mass ratio of the nucleic acid to the transfection reagent can be 1:(1 - 200).

[0054] In some embodiments, the mixing can be carried out at room temperature.

[0055] In some embodiments, the mixing time may be 0.1 to 20 min, for example 1 min.

[0056] The embodiment of the present invention provides a method for safely and efficiently transfecting nucleic acids in suspended cells, comprising: using the aforementioned method to prepare a transfection reagent with a suspended cell-specific substance and a nucleic acid delivery polymer, and using the transfection reagent to deliver nucleic acids into suspended cells.

[0057] Another aspect of the embodiments of the present invention further provides the use of the aforementioned transfection reagent to efficiently deliver nucleic acids in suspended cells. The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0058] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0059] Example 1 This embodiment provides a transfection reagent that can be used for suspension cells and is used to transfect GFP-mRNA into MOLM13 cells (suspension cells). MOLM13 cells are a human acute myeloid leukemia cell line. Specifically, the process includes the following steps: (1) Add terephthalaldehyde (10 mg / mL) and PBAE (100 mg / mL) into 50 μL tetrahydrofuran (THF) at a volume ratio of 1:1; (2) In an ultrasonic environment, the above solution is added to 500 μL of pure water in small amounts for purification to obtain the transfection reagent. In this step, other mixing methods can also be used, such as ultrasonic emulsification, thin film dispersion, mechanical stirring, and microfluidics, which can achieve comparable technical effects.

[0060] (3) The sample prepared in step (2) is concentrated by ultrafiltration using a 100K ultrafiltration tube to obtain transfection reagent-1.

[0061] (4) The transfection reagent-1 obtained in step (3) was co-incubated with GFP-mRNA (purchased from Anshengda) to transfect MOLM13 cells: 3×10 4300 μL of MOLM13 cells were cultured for 24 hours; 900 ng of mRNA (GenBank: LN515608.1) was mixed with 15 μL of sodium acetate and incubated for 5 min; 15 μL of transfection reagent-1 was mixed with 15 μL of sodium acetate and incubated for 5 min; then the mixed solution of GFP-mRNA was added to the mixed solution of the transfection reagent, and they were co-incubated at room temperature for 5 min; 150 μL of opti-MEM medium (Thermo Fisher) was added and mixed well. Finally, the mixed solution was added to the cell culture plate. After three hours, the medium was aspirated and replaced with fresh medium. After culturing for 12 hours, the cells were observed and photographed with an inverted fluorescence microscope, or the transfection effect of the cells was detected by flow cytometry, or the cell viability was detected.

[0062] Refer to Figure 2 , which is the transmission electron microscopy image of the nucleic acid loaded by transfection reagent-1 prepared in this example; as can be seen from the figure, the particle size of the transfection reagent is about 50 nm.

[0063] Figure 3 This is the comparison diagram of the tracer labeling of MOLM13 cells after transfection reagent-1 prepared in this example and Lipo6000 in the prior art were co-incubated with mRNA expressing green fluorescent protein (GFP-mRNA) respectively, and the comparison diagram of the flow cytometry quantitative results. As can be seen from the figure, under the same transfection conditions, when transfecting MOLM13 cells, the expression level of MOLM13 cells after transfection with transfection reagent-1 is significantly higher than that of Lipo6000. The transfection efficiency of transfection reagent-1 is as high as 64.23%, while the transfection efficiency of Lipo6000 is only 1.64%; obviously, after transfection with transfection reagent-1, the labeling signal in the cells is significantly better than that of Lipo6000.

[0064] Obviously, the transfection reagent provided in this example can be used for the transfection of suspension cells and achieve high-efficiency nucleic acid delivery of suspension cells.

[0065] Example 2 In this example, a transfection reagent was prepared by the following method and used to transfect mCherry plasmid (Beyotime: D2711) into hematopoietic stem cells (suspension cells), which is expected to be used for gene editing of hematopoietic stem cells. The steps include: (1) Palmitic anhydride and PBAE (100 mg / mL) were added to 100 μL of DMSO at a volume ratio of 1:2. (2) In an ultrasonic environment, the above solution was added to 1 mL of pure water in small amounts multiple times for purification to obtain the transfection reagent.

[0066] (3) The sample prepared in step (2) was ultrafiltered and concentrated with a 100K ultrafiltration tube to obtain transfection reagent-2.

[0067] (4) Mix the transfection reagent obtained in step (3) with the mCherry plasmid (for 10 min) and transfect it into hematopoietic stem cells.

[0068] Refer to Figure 4 , which is a columnar comparison chart of the expression levels of mCherry protein after transfecting the mCherry plasmid with the transfection reagent - 2 prepared in this example and Lipo6000 in the prior art respectively. As can be seen from the figure, under the same transfection conditions, when transfecting the mCherry plasmid, the expression level of mCherry protein after transfection with the transfection reagent - 2 is significantly higher than that of Lipo6000. The expression level of mCherry protein transfected with the transfection reagent - 2 is greater than 1.0×10 5 a.u., while the expression level of mCherry protein transfected with Lipo6000 is less than 3.0×10 4 a.u.

[0069] In the prior art, sickle cell disease patients' hematopoietic stem cells are usually corrected by the CRISPR - Cas9 gene editing technology, and then normal hemoglobin is regenerated to replace the defective hemoglobin to treat sickle cell disease. However, directly introducing the CRISPR - Cas9 gene into hematopoietic stem cells is still challenging. However, from the above results, it can be seen that the transfection reagent provided by the present invention can be used to transfect the mCherry plasmid into hematopoietic stem cells, and it is expected to be used for hematopoietic stem cell gene editing to treat sickle cell disease.

[0070] Example 3 In this example, a transfection reagent was prepared by the following method and used to transfect the CAR plasmid (Addgene: 113014) into NK cells. The steps include: (1) Add N - succinimidyl myristate (10 mg / mL) and PBAE (100 mg / mL) to 300 μL of DMSO in a volume ratio of 2:1; (2) In an ultrasonic environment, add the above solution to 10 mL of pure water in small amounts multiple times for purification treatment to obtain the transfection reagent.

[0071] (3) Ultrafilter and concentrate the sample prepared in step (2) with a 100K ultrafiltration tube to obtain transfection reagent - 3.

[0072] (4) Mix the transfection reagent obtained in step (3) with the CAR plasmid (for 10 min) and transfect it into NK cells.

[0073] Refer to Figure 5, for the transfection reagent - 3 of this example and the blank control (cultured in opti - MEM), the NK cells transfected with the CAR plasmid were used for cell activity control. As can be seen from the figure, the transfection process has almost no effect on cell activity, indicating that the nucleic acid transfection reagent has good biocompatibility.

[0074] Chimeric antigen receptor NK cell immunotherapy (CAR - NK) is similar to CAR - T cell therapy. It delivers the CAR plasmid into NK cells through viral technology and has entered the clinical trial stage. However, delivering the CAR plasmid into NK cells through non - viral technology is still challenging, mainly due to the toxic effect of non - viral delivery vectors on cells. The transfection reagent provided by the present invention can be used to transfect the CAR plasmid into NK cells with almost no effect on the activity of NK cells, indicating that the nucleic acid transfection reagent provided by the present invention meets the biocompatibility requirements of CAR - NK and is expected to be applied to the preparation and clinical practice of CAR - NK.

[0075] Example 4 The difference between this example and Example 1 is that terephthalaldehyde in step (1) is replaced with 10 mg / mL dopamine hydrochloride, which is the transfection reagent - 4 of this example. Refer to Figure 6 。

[0076] Example 5 The difference between this example and Example 1 is that terephthalaldehyde in step (1) is replaced with 10 mg / mL dopamine hydrochloride, which is the transfection reagent - 5 of this example. Refer to Figure 6 。

[0077] Example 6 The difference between this example and Example 1 is that terephthalaldehyde in step (1) is replaced with 10 mg / mL 3,3'-dithiobis(propionyl hydrazide), which is the transfection reagent - 6 of this example. Refer to Figure 6 。

[0078] Example 7 The difference between this example and Example 1 is that terephthalaldehyde in step (1) is replaced with 10 mg / mL 1-(3 - mercapto - 1 - oxopropoxy)-2,5 - pyrrolidinedione, which is the transfection reagent - 7 of this example. Refer to Figure 6 。

[0079] Example 8 The difference between this example and Example 1 is that terephthalaldehyde in step (1) is replaced with 10 mg / mL DOTMA, which is the transfection reagent - 8 of this example. Refer to Figure 6 。

[0080] Example 9 The difference between this example and Example 1 is that terephthalaldehyde in step (1) is replaced with 10 mg / mL N-hydroxysuccinimide palmitate, which is the transfection reagent-9 of this example. Refer to Figure 6 .

[0081] Example 10 The difference between this example and Example 1 is that terephthalaldehyde in step (1) is replaced with 10 mg / mL sodium palmitate, which is the transfection reagent-10 of this example. Refer to Figure 6 .

[0082] As shown by Figure 6 , after the transfection reagents prepared in Examples 4-10 were co-incubated with mRNA expressing green fluorescent protein (GFP-mRNA) and then transfected into MOLM13 cells, the comparative diagrams of the tracer labels of the cells were obtained after 12 hours. It can be seen from the figure that under the same transfection conditions, when transfecting MOLM13 cells, the expression level of MOLM13 cells after transfection with transfection reagent-1 was significantly higher than that of Lipo6000 ( Figure 3 ).

[0083] Obviously, the nucleic acid transfection positive rate of the transfection reagent of the present invention in suspension cells (such as GFP-mRNA, mCherry, CAR plasmid, etc., positive rate > 80%) is significantly greater than that of Lipo6000 (a product of Beyotime, product number: C0526FT) in suspension cells (1.64%), and it has excellent biocompatibility.

[0084] In summary, the nucleic acid transfection reagent prepared by the technical solution of the present invention can not only achieve efficient transfection of suspension cells and overcome the difficulty of difficult transfection of suspension cells, but also avoid the toxic effect of the nucleic acid delivery by using a non-viral nucleic acid delivery vector (cationic liposome) on cells, realize the nucleic acid delivery of the non-viral nucleic acid delivery vector, and has a high cell viability and excellent biocompatibility.

[0085] Effective delivery of nucleic acids and high positive rate expression are achieved in suspension cells, overcoming the difficulties of existing transfection reagents in efficiently transfecting and efficiently delivering nucleic acids. Moreover, the nucleic acid transfection reagent also has good biocompatibility and can be used for function enhancement and tracing of suspension cells.

[0086] In addition, the inventors of this case also referred to the foregoing examples and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0087] Aspects, embodiments, features and examples of the present invention should be considered illustrative in all respects and not limiting of the present invention, the scope of which is defined by the claims. Without departing from the spirit and scope of the claimed invention, those skilled in the art will appreciate other embodiments, modifications and uses.

[0088] The use of the title of the present invention does not imply a limitation of the present invention; the Summary of the Invention and the Detailed Description sections may apply to any aspect, embodiment or feature of the present invention.

[0089] Throughout the present invention, where a composition is described as having, comprising or including a particular component or where a process is described as having, comprising or including a particular process step, it is contemplated that the compositions of the present invention are also essentially composed of or consist of the recited components, and the processes of the present invention are also essentially composed of or consist of the recited process steps.

[0090] Unless specifically stated otherwise, the use of the terms “include (include, includes, including)”, “have (have, has or having)” should generally be understood to be open-ended and non-limiting.

[0091] It should be understood that the order of the steps or the order of performing a particular action is not of great importance as long as the teachings of the present invention remain operable. In addition, two or more steps or actions may be performed simultaneously.

[0092] The above are only the preferred embodiments of the present invention, and do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any changes, modifications, substitutions, integrations and parameter changes made to these embodiments within the spirit and principle of the present invention, by means of conventional substitutions or capable of achieving the same functions without departing from the principle and spirit of the present invention, fall within the protection scope of the present invention.

Claims

1. A nucleic acid transfection reagent for suspension cells, characterized in that, The nucleic acid transfection reagent is a nanoparticle assembled from a nucleic acid delivery polymer and a suspension cell-specific substance; wherein, The surface of the nucleic acid transfection reagent is positively charged, and can adsorb negatively charged nucleic acids onto the surface of the nanoparticle through electrostatic interaction to achieve the transportation of nucleic acids; The suspension cell-specific substance can bind to the surface characteristic groups of suspension cells to achieve nucleic acid transfection; The nucleic acid transfection reagent can load nucleic acids to transfect suspension cells to achieve the delivery or expression of nucleic acids in suspension cells; The positive charge comes from the nucleic acid delivery polymer.

2. The nucleic acid transfection reagent for suspended cells according to claim 1, wherein The suspension cell-specific substance includes at least one of lauric aldehyde, dopamine hydrochloride, stearic acid N-hydroxysuccinimide ester, 2,5-dioxopyrrolidin-1-yl myristate, palmitic acid hydrazide, palmitic anhydride, dodecyl aldehyde, N,N'-dicyclohexylcarbodiimide, 3,3'-dithiobis(propionyl hydrazide), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-succinimidyl myristate, terephthalaldehyde, sodium palmitate, palmitic acid N-hydroxysuccinimide ester, DOTMA lipid, disuccinimidyl suberate, 1-(3-mercapto-1-oxopropoxy)-2,5-pyrrolidinedione.

3. The nucleic acid transfection reagent for suspension cells according to claim 1, wherein The nucleic acid delivery polymer is poly-β-amino ester; the molecular weight is 100~200000 Da; The nucleic acid delivery polymer is slightly soluble in water, has lipophilicity, and has nucleic acid binding sites; and / or, the nanoparticle is a nanomicelle or vesicle; and / or, the nanoparticle is prepared by any one of ultrasonic dispersion method, ultrasonic emulsification method, thin film dispersion method, mechanical stirring method and microfluidic method; and / or, the nucleic acid delivery polymer and the suspension cell-specific substance are assembled into the nanoparticle by hydrophobic interaction at a volume ratio of 1:1~20; and / or, the nucleic acid includes one of small molecule messenger nucleic acid, mRNA, reporter gene mRNA, therapeutic gene mRNA, interfering gene plasmid, therapeutic protein nucleic acid plasmid and reporter gene plasmid, dsRNA, antisense nucleic acid, antisense oligonucleotide, microRNA, antisense microRNA, microRNA inhibitor, microRNA activator and immunostimulatory nucleic acid.

4. A method for preparing a nucleic acid transfection reagent for suspending cells according to any one of claims 1-3, characterized in that, The nucleic acid transfection reagent is prepared by mixing a nucleic acid delivery polymer and a suspension cell-specific substance; specifically includes the following steps: S1. Add the suspension cell-specific substance and poly-β-amino ester into a solvent and mix to obtain a mixed solution; S2. In an ultrasonic environment, add the mixed solution into pure water and mix evenly, and perform purification treatment to obtain the nucleic acid transfection reagent.

5. According to the preparation method described in claim 4, wherein, In S1, in the mixed solution, the mass ratio of the poly-β-amino ester to the suspension cell-specific substance is 1:(5~200); and / or, in S2, the solvent is any one of ethanol, dichloromethane, chloroform, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide; The mixing method of the mixed solution and the aqueous solution includes any one of ultrasonic emulsification method, thin film dispersion method, mechanical stirring method and microfluidic method; The purification treatment is ultrafiltration purification treatment.

6. A method for transfecting nucleic acid into suspension cells, comprising using the nucleic acid transfection reagent according to any one of claims 1-4 as a nano-delivery vector, co-incubating the nucleic acid transfection reagent with nucleic acid, then adding the mixture to suspension cells, mixing evenly, and then culturing to deliver the nucleic acid into the suspension cells, thereby achieving nucleic acid transfection.

7. The method for transfecting nucleic acid into suspended cells according to claim 6, characterized in that, The specific steps include: first mixing nucleic acid with sodium acetate and incubating for 2-20 min to obtain a nucleic acid mixture; Adding the nucleic acid mixture to the nucleic acid transfection reagent mixture, mixing evenly, and then co-incubating for 2-20 min to obtain a transfection mixture; Adding the transfection mixture to a culture medium and mixing well to form a transfection culture solution; Finally, replacing the suspension cell culture medium with the transfection culture solution and culturing for 1-24 hours to complete the nucleic acid transfection of the suspension cells; And / or, the mass ratio of the nucleic acid to the transfection reagent can be 1:(1-200).

8. Use of the nucleic acid transfection reagent according to any one of claims 1-3 in transfection or labeled tracing of suspension cells.

9. Use of the nucleic acid transfection reagent according to any one of claims 1-3 in delivering nucleic acid to hematopoietic stem cells and NK cells.

10. Use of the nucleic acid transfection reagent according to any one of claims 1-3 in non-virally delivering a CAR plasmid into NK cells as a delivery vector.

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