Preparation method and application of transfection compound
By using microfluidic equipment to mix and optimize the ratio of transfection reagents and plasmids during the preparation of transfection complexes, the problem of uneven particle size distribution of complexes is solved, and the transfection efficiency is significantly improved.
Patent Information
- Application Number
- CN202311247450.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-25
- Publication Date
- 2025-03-25
AI Technical Summary
The existing methods for preparing transfected complexes result in uneven distribution of complex particle size, high aggregation and low transfection efficiency.
The transfection reagent and the plasmid to be transfected were mixed and processed using microfluidic control equipment. By optimizing the dilution ratio of the transfection reagent and the concentration of the plasmid, a transfection complex with uniform particle size was prepared.
The particle size uniformity and transfection efficiency of the transfection complex are improved, allowing the plasmid to enter the target cells more efficiently.
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Figure HDA0004469158320000011 
Figure HDA0004469158320000012
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of in vitro cell gene transfection, and specifically to a method for preparing a transfection complex and its application. Background Art
[0002] Virus packaging is a key step in the field of biomedical research and gene therapy. It involves inserting the target gene into a viral vector and preparing viral particles in host cells for gene delivery and expression. In order to successfully carry out viral packaging, efficient transfection reagents are required to ensure that plasmids (including vector plasmids, envelope plasmids, packaging plasmids, etc.) are co-transfected in host cells to form a fully functional viral vector.
[0003] Traditionally, cationic liposomes are one of the commonly used transfection reagents. They have a positively charged lipid structure that can attract the negative charge of the plasmid and promote the endogenous absorption of the plasmid. Another commonly used transfection reagent is PEI cationic polymer, which has excellent transfection efficiency and stability and is widely used in the field of virus packaging and gene transfection.
[0004] The preparation process of transfection complex usually involves mixing a small volume of vector solution with a large volume of plasmid solution, achieving uniform mixing by inverting the container, and then incubating the mixture to form an efficient viral vector. However, existing methods for preparing transfection complexes have problems such as low transfection efficiency.
[0005] To this end, the present application proposes a novel method for preparing a transfection complex. Summary of the invention
[0006] This application is completed by the inventor based on the following findings:
[0007] At present, manual mixing is usually used in the preparation of transfection complexes. However, this step often leads to uneven particle size distribution of the complex and a high degree of aggregation (PDI>0.35). According to the dynamic light scattering particle size analysis standard ISO 22412:2017, when the PDI value is less than 0.1, the sample is considered to be monodisperse. However, due to the limitations of the manual mixing method, the dispersion of the complex is poor and does not meet the monodispersity requirements.
[0008] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, one purpose of the present application is to propose a method for preparing a transfection complex with high transfection efficiency.
[0009] In the first aspect of the present application, the present application proposes a method for preparing a transfection complex. According to an embodiment of the present application, the method comprises: using a microfluidic device to perform a first mixing treatment on a transfection reagent and a plasmid to be transfected, so as to obtain a transfection complex; wherein the mass volume ratio of the plasmid to be transfected to the transfection reagent is 1 mg: 1 mL to 1 mg: 3 mL.
[0010] According to the embodiments of the present application, the transfection complex prepared by the present method has better particle size uniformity and higher transfection efficiency.
[0011] According to an embodiment of the present application, the above method further includes at least one of the following technical features:
[0012] According to an embodiment of the present application, the transfection reagent is diluted with a serum-free medium. Since the transfection reagent generally contains cationic compounds, such as PEI (polyethyleneimine), etc., it has certain cytotoxicity. By mixing and diluting the transfection reagent with a serum-free medium, the toxicity of the reagent to cells can be reduced, reducing cell damage.
[0013] According to an embodiment of the present application, the dilution ratio of the transfection reagent in the serum-free medium is 1:20 to 1:40. In some examples of the present application, the dilution ratio of the transfection reagent in the serum-free medium is optionally 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40.
[0014] According to an embodiment of the present application, the dilution ratio of the transfection reagent in the serum-free medium is 1:25. The inventors have found through a large number of experimental verifications that when the dilution ratio of the transfection reagent in the serum-free medium is 1:25, it has a higher transfection efficiency.
[0015] According to an embodiment of the present application, the plasmid is provided in a form dissolved in a serum-free medium.
[0016] According to an embodiment of the present application, the concentration of the plasmid in the serum-free medium is 0.02-0.06 mg / mL. In some examples of the present application, the concentration of the plasmid in the serum-free medium is optionally 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL.
[0017] According to an embodiment of the present application, the concentration of the plasmid in the serum-free medium is 0.04 mg / mL. The concentration of the plasmid directly affects the ability of the plasmid DNA to enter the target cells, thereby affecting the transfection efficiency. The inventors have verified through a large number of experiments that the plasmid has a higher transfection efficiency when the concentration in the serum-free medium is 0.04 mg / mL.
[0018] According to the embodiments of the present application, the microfluidic device parameters include: a flow rate of 18 ml / min, a total flow rate of 1.2 to 12 ml, a flow rate of 0.35 to 0.9 ml, and a waste liquid of 0.2 to 0.4 ml. In some examples of the present application, the inventors conducted gradient screening on the total flow rate (1.2 ml, 1.5 ml, 3 ml, 4.5 ml, 6 ml, 7.5 ml, 9 ml, 12 ml), the front waste liquid (0.35 ml, 0.5 ml, 0.75 ml, 0.9 ml), and the rear waste liquid (0.2 ml, 0.3 ml, 0.4 ml) of the microfluidic device, and finally found that within the range of a flow rate of 18 ml / min, a total flow rate of 1.2 to 12 ml, a flow rate of 0.35 to 0.9 ml, and a waste liquid of 0.2 to 0.4 ml, a transfection complex with uniform particle size can be obtained.
[0019] According to an embodiment of the present application, the mass volume ratio of the plasmid to the transfection reagent is 1 mg: 1 mL to 1 mg: 1 mL. In some preferred examples of the present application, when the mass volume ratio of the plasmid to the transfection reagent is 1 mg: 1 mL to 1 mg: 1 mL, the transfection efficiency is the highest.
[0020] According to an embodiment of the present application, the transfection reagent is polyethyleneimine (PEI).
[0021] According to the embodiments of the present application, the particle size of the transfection complex is no greater than 100 nm. In some examples of the present application, the transfection complex needs to be transported to the cell nucleus for expression. The cell nuclear pore is about 100 nm, and transfection complexes with a similar particle size can carry more plasmids and carry the plasmids into the cell nucleus smoothly. Transfection complexes larger than 100 nm cannot pass through the nuclear pore.
[0022] According to an embodiment of the present application, the plasmid to be transfected is selected from at least one of PSPAX2, PMD2.G and CAPN3.
[0023] In the second aspect of the present application, the present application proposes a transfection complex. According to an embodiment of the present application, the transfection complex is prepared by the method described in the first aspect or any embodiment described in the first aspect of the present application. According to an embodiment of the present application, the transfection complex has a uniform particle size and good transfection efficiency.
[0024] In the third aspect of the present application, the present application proposes a method for transfecting cells. According to an embodiment of the present application, the method comprises using the transfection complex described in the second aspect of the present application to transfect the cells to be transfected. According to an embodiment of the present application, the method can achieve efficient transfection of cells.
[0025] According to an embodiment of the present application, the method for transfecting cells further includes at least one of the following technical features:
[0026] According to an embodiment of the present application, before the transfection treatment, the transfection complex is further allowed to stand at room temperature for 10 to 20 minutes. In some examples of the present application, the transfection reagent and the plasmid are allowed to stand to interact and form a stable transfection complex, thereby improving the transfection efficiency.
[0027] In the fourth aspect of the present application, the present application proposes a method for harvesting viruses. According to an embodiment of the present application, the method comprises: subjecting the transfection complex described in the second aspect of the present application to transfection of the cells to be transfected; culturing the transfected cells under conditions suitable for cell growth to harvest the viruses. According to an embodiment of the present application, harvesting viruses by transfecting cells with the transfection complex can increase the virus yield.
[0028] According to an embodiment of the present application, the method for harvesting viruses further includes at least one of the following technical features:
[0029] According to an embodiment of the present application, the culture treatment is carried out at 25° C. and 5% CO 2 .
[0030] According to an embodiment of the present application, the culture treatment time is 72 to 96 hours. In some examples of the present application, the culture treatment time can be adjusted based on experimental requirements.
[0031] It should be noted that in the present application, the features and advantages described for the methods, devices and systems in any of the above aspects, implementation modes or examples are also applicable to other aspects and will not be repeated here.
[0032] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:
[0034] Figure 1This is a schematic diagram of the verification result of the encapsulation efficiency of the PEI vector transfection complex according to one embodiment of the present application; wherein the left side is a plasmid molecular weight standard, the middle is a transfection complex, and the right side is a plasmid mixture to be transfected;
[0035] Figure 2 It is a schematic diagram of the comparison results of transfection efficiency of PEI complex prepared by manual mixing and microfluidics according to an embodiment of the present application.
[0036] The above drawings are only schematic and have no limiting effect. In the drawings, for illustrative purposes, the sizes of some components may be exaggerated and not drawn to scale. The sizes and relative sizes do not necessarily correspond to the actual restoration when the present invention is implemented. DETAILED DESCRIPTION
[0037] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.
[0038] As used herein, the singular forms "a", "an", etc. include plural referents (one or more). "A set" or "a plurality" refers to two or more.
[0039] In this document, "include" or "comprising" is an open expression, including the content or circumstances specified or exemplified thereafter, and also including content or circumstances that are applicable or consistent with the stated circumstances but are not specifically listed.
[0040] In the existing preparation process of transfection complexes, there is no report on the use of microfluidic devices to prepare transfection complexes. At present, in the preparation process of PEI transfection complexes, there is a problem of large particle size dispersion, resulting in low transfection efficiency. The inventor first proposed to use microfluidic devices to mix the transfection reagent and the plasmid to be transfected. After a large number of experimental verifications, it was found that it has good performance in improving the particle size uniformity of the transfection complex of the PEI cationic polymer carrier and enhancing the transfection efficiency.
[0041] In some examples of the present application, the inventors optimized the dilution ratio of the transfection reagent in the serum-free culture medium. The results showed that the transfection complex can be formed when the dilution ratio of the transfection reagent in the serum-free culture medium is in the range of 1:20 to 1:40, and the transfection efficiency is highest when the dilution ratio is 1:25.
[0042] In some examples of the present application, the inventors optimized the concentration of the plasmid in the serum-free culture medium accordingly. The results showed that the plasmid had a good transfection efficiency when the concentration in the serum-free culture medium was in the range of 0.02 to 0.06 mg / mL, and the transfection efficiency was highest when the concentration of the plasmid in the serum-free culture medium was 0.04 mg / mL.
[0043] In some examples of the present application, the inventors have found through experiments that the scheme of the present application can be applied to different plasmid transfections, among which the transfection effect in plasmids PSPAX2, PMD2.G and CAPN3 is better.
[0044] In some examples of the present application, the inventors found in experiments that the mass-to-volume ratio of plasmid to transfection reagent has good transfection efficiency in the range of 1 mg:1 mL to 1 mg:3 mL, among which the transfection efficiency is highest when the mass-to-volume ratio of plasmid to transfection reagent is 1 mg:1 mL.
[0045] In addition, the inventors also tried other transfection reagents, such as Lipofectamine series transfection reagents, and finally found that this solution has a better effect on PEI transfection reagents.
[0046] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.
[0047] Example 1: Preparation of transfection complex
[0048] In this example, a microfluidic device and manual mixing were used to prepare the transfection complex, and the particle size and transfection efficiency were used as evaluation indicators to assess the quality of the transfection complex.
[0049] Experimental group: Prepare transfection complex according to the specified ratio (1:1) of plasmid (μg) / transfection reagent (μl) in the instruction manual of PEI transfection kit (manufacturer: Yisheng Biology; catalog number: HB220930). Dilute 40μg plasmid PSPAX2 / PMD2.G / CAPN3 (1:1:2) with 1.0ml serum-free medium Opti-MEM, and dilute 40μl transfection reagent with 1.0ml serum-free medium Opti-MEM. Add the reagent diluent to the plasmid solution, vortex gently to mix, and let stand at room temperature for 10-20min to form a plasmid-PEI complex. Confirm that the plasmid can be completely encapsulated by electrophoresis. Electrophoresis parameters: sample amount 0.1μg (5μl), voltage 160V, agarose gel concentration 1%, electrophoresis time 30min. Take 50μl of the complex, and its average particle size is about 100nm by DLS method, and synthesize the transfection complex in the same ratio (1:1) using a microfluidic device.
[0050] Control group: Transfection complexes were prepared by manual mixing in equal proportions according to the above method.
[0051] Experimental results:
[0052] 1. The particle size characterization results in Table 1 show that there is no significant difference in particle size and particle size distribution index (PDI) between cationic liposome transfection complexes prepared manually and using microfluidic devices.
[0053] Table 1: Particle size characterization of cationic liposome transfection complexes
[0054] Preparation method Particle size (nm) PDI Manual preparation 1618.72 0.223 Microfluidic preparation 1590.06 0.223
[0055] 2. The results of particle size characterization of PEI vector transfection complexes showed that the particle size uniformity of microfluidic prepared complexes was better than that of manual mixing.
[0056] Example 2: PEI vector transfection complex preparation ratio and microfluidic process optimization
[0057] This example intends to further increase the particle size uniformity and transfection efficiency by optimizing the formulation ratio of the PEI vector transfection complex and the microfluidic process.
[0058] 1. Optimization of the preparation ratio of PEI vector transfection complex
[0059] Dilute 40 μg of plasmid PSPAX2 / PMD2.G / CAPN3 (1:1:2) with 1.0 ml serum-free medium Opti-MEM, and dilute 40 μl of transfection reagent with 1.0 ml serum-free medium Opti-MEM. Add the reagent diluent to the plasmid solution, vortex gently to mix, and let stand at room temperature for 10-20 minutes to form a plasmid-PEI complex. Confirm the encapsulation efficiency by electrophoresis. The results are as follows Figure 1As shown, there is no free plasmid band in the transfection complex lane, indicating that the plasmid is completely encapsulated by the vector under the condition of a plasmid (μg) / vector (μl) ratio of 1:1.
[0060] 2. Microfluidic process optimization
[0061] The total flow rate was optimized under the minimum preparation volume condition (13.0ml≥total flow rate of the equipment≥1.2ml). The optimized flow rates were 6ml / min, 12ml / min, and 18ml / min respectively.
[0062] 2.1 Microfluidic preparation
[0063] The microfluidic device parameters were set to flow rate 6 ml / min, total flow 1.2 ml, front waste liquid 0.35 ml, and rear waste liquid 0.2 ml. The results are shown in Table 2.
[0064] Table 2
[0065] Particle size (nm) PDI D10(nm) D50(nm) D90(nm) 101.90 0.148 45.72 101.90 139.75 102.47 0.152 44.96 102.47 142.48 103.21 0.154 44.28 103.21 144.56
[0066] The microfluidic device parameters were set to flow rate 12 ml / min, total flow 1.2 ml, front waste liquid 0.35 ml, and rear waste liquid 0.2 ml. The results are shown in Table 3.
[0067] Table 3
[0068] Particle size (nm) PDI D10(nm) D50(nm) D90(nm) 102.04 0.131 45.35 102.04 133.31 103.59 0.125 44.84 103.59 130.16 101.92 0.137 44.01 101.92 134.84
[0069] The microfluidic device parameters were set to flow rate 18 ml / min, total flow 1.2 ml, front waste liquid 0.35 ml, and rear waste liquid 0.2 ml. The results are shown in Table 4.
[0070] Table 4
[0071] Particle size (nm) PDI D10(nm) D50(nm) D90(nm) 103.42 0.092 45.81 103.42 132.48 102.57 0.095 45.03 102.57 133.75 105.22 0.088 46.83 105.22 134.26
[0072] The microfluidic device parameters were set to flow rate 18 ml / min, total flow 6.0 ml, front waste liquid 0.5 ml, and rear waste liquid 0.2 ml. The results are shown in Table 5.
[0073] Table 5
[0074] Particle size (nm) PDI D10(nm) D50(nm) D90(nm) 104.68 0.095 45.81 103.42 135.21 103.01 0.090 45.97 102.57 131.42 105.84 0.091 46.29 105.22 132.59
[0075] The microfluidic device parameters were set to flow rate 18 ml / min, total flow 12.0 ml, front waste liquid 0.9 ml, and rear waste liquid 0.4 ml. The results are shown in Table 6.
[0076] Table 6
[0077] Particle size (nm) PDI D10(nm) D50(nm) D90(nm) 103.25 0.096 45.23 103.25 135.26 103.91 0.089 47.16 103.91 133.38 104.16 0.090 45.15 104.16 130.01
[0078] From the results in Tables 2 to 6, it can be concluded that under different flow rate conditions, the particle size uniformity of the PEI transfection complex prepared by microfluidics is good and it is a monodisperse system.
[0079] 2.2 Manual mixing
[0080] The results are shown in Table 7. It shows that the particle size uniformity of the PEI mixture prepared by manual mixing is poor and it is not a monodisperse system.
[0081] Table 7
[0082] Particle size (nm) PDI D10(nm) D50(nm) D90(nm) 103.01 0.364 46.38 103.01 302.18 105.69 0.372 46.66 105.69 314.28 103.97 0.376 45.78 103.97 317.46
[0083] 3. Comparison of transfection efficiency
[0084] 2.5 × 10 cells were seeded in a 10 cm dish. 6 293T cells were cultured and transfection complex was prepared 24 hours later for transfection. Transfection complex preparation: 40 μg plasmid PSPAX2 / PMD2.G / CAPN3 (1:1:2) was diluted with 1.0 ml serum-free medium Opti-MEM, and 40 μl transfection reagent was diluted with 1.0 ml serum-free medium Opti-MEM. Add the reagent diluent to the plasmid solution, vortex gently to mix, and let stand at room temperature for 10-20 minutes to form a plasmid-PEI complex. Add 1.0 ml of the complex to 10 ml of cultured cells. Observe the expression of green fluorescent protein after 48 hours. The dilution ratio of transfection reagent and plasmid in serum-free medium, as well as the volume ratio of plasmid to transfection reagent were all in accordance with the instructions and were not optimized.
[0085] The results are as follows Figure 2 As shown, the transfection complex prepared by the microfluidic chip has high particle size uniformity and better transfection efficiency than manual mixing.
[0086] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.
[0087] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.
Claims
1. A method for preparing a transfection complex, characterized in that: include: Using a microfluidic device to perform a first mixing process on a transfection reagent and a plasmid to be transfected, so as to obtain a transfection complex; Wherein, the mass volume ratio of the plasmid to be transfected to the transfection reagent is 1 mg:1 mL to 1 mg:3 mL.
2. The method according to claim 1, characterized in that The transfection reagent is diluted with serum-free medium; Optionally, the dilution ratio of the transfection reagent in the serum-free medium is 1:20 to 1:40, preferably 1:
25.
3. The method according to claim 1, characterized in that The plasmid is provided in the form of being dissolved in serum-free medium; Optionally, the concentration of the plasmid in the serum-free medium is 0.02-0.06 mg / mL, preferably 0.04 mg / mL.
4. The method according to claim 1, characterized in that: The microfluidic device parameters include: flow rate of 18 ml / min, total flow of 1.2-12 ml, front waste liquid of 0.35-0.9 ml, and rear waste liquid of 0.2-0.4 ml.
5. The method according to claim 1, characterized in that The mass volume ratio of the plasmid to the transfection reagent is 1 mg:1 mL to 1 mg:1 mL.
6. The method according to claim 1, characterized in that The transfection reagent is polyethyleneimine.
7. The method according to claim 1, characterized in that The particle size of the transfection complex is no greater than 100 nm.
8. The method according to claim 1, characterized in that The plasmid to be transfected is selected from at least one of PSPAX2, PMD2.G and CAPN3.
9. A transfection complex, characterized in that The transfection complex is prepared by the method according to any one of claims 1 to 8.
10. A method for transfecting cells, characterized in that: The transfection complex according to claim 7 is used to perform transfection treatment on the cells to be transfected.
11. The method according to claim 10, characterized in that Before the transfection treatment, the method further comprises allowing the transfection complex to stand at room temperature for 10 to 20 minutes.
12. A method for harvesting viruses, characterized in that: include: Transfecting the cells to be transfected with the transfection complex according to claim 7; The transfected cells are cultured under conditions suitable for cell growth to harvest the virus.
13. The method according to claim 12, characterized in that The culture treatment is carried out at 25°C and 5% CO2; Optionally, the culture treatment time is 72 to 96 hours.