Gene transfection vector based on dendritic polyamidoamine as well as preparation method and application of gene transfection vector

Abstract

The invention discloses a gene transfection vector based on dendritic polyamidoamine, which has good water solubility and biocompatibility, and can load exogenous nucleic acid molecules in a water environment to form a stable gene transfection vector compound, so that the gene transfection vector can be used as a nucleic acid vector to carry the exogenous nucleic acid molecules to enter cells. The invention also discloses a preparation method and application of the carrier. The preparation method is simple and efficient, the prepared carrier is applied to the field of agricultural pests, pest control can be performed by loading exogenous nucleic acid, and harm of pests to plants can be greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical fields of polymer chemistry, biomaterials and agricultural pest control, and specifically relates to a gene transfection vector based on dendritic polyamidoamine, and a preparation method and application thereof. Background Art

[0002] Pest control in my country faces problems such as pesticide resistance, residues and food safety. The strategy of using double-stranded RNA (dsRNA) to control pests can specifically and efficiently reduce the expression of specific genes, thereby regulating the growth and development of pests and harmful behaviors, which is of great significance for efficient and green control of pests. At the same time, dsRNA insecticide molecules can be naturally degraded in the environment and are not easy to cause pests to develop resistance, making them one of the ideal sources for green pest control. However, for most pests, especially lepidopteran pests, dsRNA is absorbed by pest cells or tissues, inhibiting the insecticidal effect of dsRNA. At present, the laboratory often uses injection to introduce dsRNA insecticide molecules into the body of the pest, which is cumbersome and requires high precision instruments and technicians, limiting the marketization of dsRNA.

[0003] In recent years, nanotechnology has shown great application potential in the agricultural field. The targeted transmission and controlled release functions of nanocarriers are used to deliver nucleic acid drugs dsRNA into insects, breaking the barriers of the organ basement membrane, cell membrane and intestinal peritrophic membrane in the insects, promoting the absorption of exogenous nucleic acid molecules by cells, and improving the efficiency of exogenous nucleic acid molecules. Therefore, the nucleic acid introduction system developed based on nanocarriers is expected to improve the efficiency of dsRNA insecticidal molecules, simplify the dsRNA introduction process, and help the marketization of the insecticidal molecules.

[0004] Dendritic polymer molecules have a large number of cavities and carry a large number of surface functional groups. They can be widely used in the field of biomedicine. In particular, polyamide-amine dendritic polymers have been widely studied and applied in gene transfection due to their high efficiency, safety and low toxicity. In order to obtain better transfection effect and biocompatibility, a gene transfection vector based on chemical covalent modification of fluoride on the surface of dendritic polymers was prepared. The fluoride modification group can stably bind nucleic acid substances, effectively protect nucleic acid substances and promote the cellular uptake of carrier / nucleic acid drug complexes, ensuring that the nucleic acid drug complexes act efficiently on intracellular targets. Therefore, this type of partially fluoride-surface-modified hyperbranched polymers has the advantages of efficient transfection, low cytotoxicity, simple synthesis method and high yield. It is particularly important to establish a pest control system for fluoride-surface-modified hyperbranched polymer nanocarriers to deliver exogenous nucleic acid drugs dsRNA, which has positive significance for the development of pesticide reduction and pest control and green pest control technology. Summary of the invention

[0005] The first technical problem to be solved by the present invention is to provide a gene transfection vector based on dendritic polymers, which has good water solubility and biocompatibility, can enter living cells, and can bind to negatively charged nucleic acid molecules dsRNA through electrostatic forces, thereby being used as a nucleic acid vector to carry exogenous nucleic acid molecules dsRNA into cells.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned carrier.

[0007] In order to solve the above-mentioned first technical problem, the gene transfection vector based on dendritic polyamidoamine provided by the present invention has the following structure: A-(NHCH2CH2SO2NHCH2CF2CF2CF3) n , the A is obtained by reacting n terminal amino groups of a dendritic polyamidoamine PAMAM with n vinylsulfonyl fluorides respectively and then with n terminal amino fluorides, the total terminal amino group of the dendritic polyamidoamine PAMAM is m, m is 32 or 64, and n is a natural number between 16 and m.

[0008] To solve the above second technical problem, the preparation method provided by the present invention comprises the following steps:

[0009] 1) Dendritic polyamidoamine PAMAM and vinylsulfonyl fluoride are mixed in a molar ratio of (1:1)-(1:40), and reacted in an organic solvent at 45-55° C. to synthesize a sulfonyl fluoride-containing dendritic polymer.

[0010] 2) A fluorinated dendritic polyamidoamine (PAMAM-F) was synthesized by a sulfonyl fluoride click polymerization reaction of a sulfonyl fluoride-containing dendritic polymer with a terminal amino fluoride (same molar amount as vinyl sulfonyl fluoride) in the presence of a catalyst at 30-50°C.

[0011] Preferably, the organic solvent is a mixed solution of methanol and water, ethanol or DMF.

[0012] Preferably, the terminal amino fluoride is heptafluorobutylamine, and the structure of the prepared fluorinated dendritic polyamidoamine (PAMAM-7F) can be as follows: (Here, the terminal amino group not bound to vinylsulfonyl fluoride is represented by only one NH2) and is represented by PAMAM-7F.

[0013] Preferably, the catalyst is 1,8-diazacycloundec-7-ene (DBU) or 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD).

[0014] The present invention also provides the use of the fluorine-containing dendritic polyamidoamine as a nucleic acid carrier at the cellular level and the living body level.

[0015] The synthesized fluorine-containing dendritic polyamidoamine (PAMAM-F) was incubated with living cells. The heptafluorine-containing dendritic polyamidoamine (PAMAM-7F) can quickly and efficiently enter the living cells, and can be combined with exogenous nucleic acid drugs such as dsRNA molecules for controlling pests as a carrier to carry exogenous nucleic acid substances to efficiently and quickly transfect living cells. The PAMAM-7F / dsRNA complex was further sprayed on the body surface of the important rice pest Chilo suppressalis in my country. PAMAM-7F / dsRNA penetrated the body wall barrier and cell membrane barrier of the pest and entered the cell, indicating that PAMAM-7F can carry dsRNA from the body surface of the Chilo suppressalis into the body of the living pest. The experiment shows that PAMAM-7F has the conditions for use as a nucleic acid carrier in vitro or in vivo.

[0016] The present invention has the following beneficial effects:

[0017] The invention has the advantages of simple synthesis, high efficiency, simple product purification, etc., and the synthesized fluorine-containing dendritic polyamidoamine (PAMAM-F) is safe, low-toxic, and has excellent water solubility. It can be used as an efficient nucleic acid carrier and applied to scientific research and green pest control in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a fluorescence image and fluorescence statistical image showing that the PAMAM-7F series vectors carrying dsRNA efficiently enter living cells of pests in Example 2 of the present invention.

[0019] Figure 2 These are the GFP fluorescence images and GFP fluorescence intensity statistical graphs after Sf9 cells were treated with dsRNA alone and different nanocarriers / dsRNA in Example 2.

[0020] Figure 3 The nanocarrier PAMAM*7F23 in Example 2 promotes the entry of dsRNA molecules into the body of pests and improves the efficiency of dsRNA molecules, thereby significantly inhibiting the damage caused by pests to plants. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the dendritic polyamidoamine PAMAM (G0-G4) was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd., and the raw materials used were all commercially available products.

[0022] Molecular weight <![CDATA[Number of NH2 groups at the end]]> G0 517 4 G1 1430 8 G2 3256 16 G3 6909 32 G4 14215 64

[0023] Example 1 Preparation of polysulfonamide containing heptafluoro dendrites

[0024] Dendritic polyamidoamine PAMAM (G3) (0.3517 g, 0.05 mmoL) and vinylsulfonyl fluoride (0.11 g, 1 mmoL) were reacted in DMF solution at 50°C to synthesize sulfonyl fluoride dendritic polymer, and then reacted with heptafluorobutylamine (0.1991 g, 1 mmoL) in TBD as catalyst and EtOH as solvent at 50°C to synthesize fluorinated dendritic polyamidoamine (PAMAM*7F20). PAMAM*7F20 was subjected to H NMR spectroscopy ( 1 H NMR), fluorine spectrum ( 19 F NMR), gel permeation chromatography (GPC) characterization. 1 H NMR (400MHz, DMSO-d6) δ7.94 (q, J=7.6, 5.5Hz, 49H), 3.11-2.97 (m, 117H), 2.73-2.58 (m,174H),2.58-2.47(m,157H),2.42(p,J=5.4,4.5Hz,69H),2.19(t,J=7.0Hz,121H). 19 F NMR (377MHz, D2O) δ -80.71 (s, 3F), -118.89 (s, 2F), -128.19 (s, 2F). The number average molecular weight (Mn) is 17649, and the molecular weight distribution (PDI) is 1.64.

[0025] Dendritic polyamidoamine PAMAM (G3) (0.3517 g, 0.05 mmoL) and vinylsulfonyl fluoride (0.1265 g, 1.15 mmoL) were reacted in ethanol solution at 50°C to synthesize sulfonyl fluoride dendritic polymer, and then reacted with heptafluorobutylamine (0.2289 g, 1.15 mmoL) in TBD as catalyst and EtOH as solvent at 50°C to synthesize fluorinated dendritic polyamidoamine (PAMAM*7F23). 1 H NMR, 19 F NMR and GPC Characterization. 1 H NMR (400MHz, DMSO-d6) δ7.94 (q, J=7.6, 5.5Hz, 56H), 3.11-2.97 (m, 131H), 2.73-2.58 (m,196H),2.58-2.47(m,176H),2.42(p,J=5.4,4.5Hz,77H),2.19(t,J=7.0Hz,136H). 19F NMR (377 MHz, D2O) δ -80.91 (s, 3F), -118.91 (s, 2F), -128.2 (s, 2F). The Mn of PAMAM*7F23 is 18409, and the PDI is 1.53.

[0026] Dendritic polyamidoamine PAMAM (G3) (0.3517 g, 0.05 mmoL) and vinylsulfonyl fluoride (0.154 g, 1.4 mmoL) were reacted in a methanol solution at 50°C to synthesize a sulfonyl fluoride dendritic polymer, and then reacted with heptafluorobutylamine (0.2787 g, 1.4 mmoL) in TBD as a catalyst and EtOH as a solvent at 50°C to synthesize a fluorinated dendritic polyamidoamine (PAMAM*7F28). 1 H NMR, 19 F NMR and GPC Characterization. 1 H NMR (400MHz, DMSO-d6) δ7.94 (q, J=7.6, 5.5Hz, 62H), 3.11-2.97 (m, 146H), 2.73-2.58 (m,218H),2.58-2.47(m,196H),2.42(q,J=5.4,4.5Hz,86H),2.19(t,J=7.0Hz,133H). 19 F NMR (377MHz, D2O) δ -80.92 (s, 3F), -118.91 (s, 2F), -128.21 (s, 2F). The Mn of PAMAM*7F28 is 19609 and the PDI is 1.88.

[0027] Example 2 Experiments on cellular and in vivo levels of vector-carrying dsRNA

[0028] The PAMAM-7F series synthesized in Example 1 was studied for carrying dsRNA insect control molecules into cells. The dsRNA was pre-labeled with a red fluorescent dye so that the dsRNA could be traced by a fluorescent microscope later. The difficulty of dsRNA pest control molecules alone to enter Lepidoptera cells is an important mechanism leading to the low efficiency of RNAi in Lepidoptera insects. In view of this mechanism, the PAMAM*7F series synthesized in Example 1 was assembled with dsRNA nucleic acid drugs to form a nanocarrier / dsRNA complex drug, and an efficient dsRNA delivery system was constructed. Under the action of PAMAM, PAMAM*7F20 and PAMAM*7F23, dsRNA can be effectively promoted to enter the Sf9 cells ( Figure 1 ), the dsRNA molecule alone was used as the control group and could not enter Sf9 cells ( Figure 1, A and A'). dsRNA molecules can be carried into cells via the vectors PAMAM, PAMAM*7F20 and PAMAM*7F23 ( Figure 1 , BD, B'-D'), indicating that this series of dendritic polymers have a certain ability to carry dsRNA nucleic acid drugs and promote the entry of nucleic acid drugs into pest cells; with the increase of 7F content, the ability to carry into cells is stronger (PAMAM*7F23>PAMAM*7F20>PAMAM) ( Figure 1 E), of which PAMAM*7F23 was the best, indicating that the number of 7F terminal groups has a great influence on the ability to carry genes. After using PAMAM, PAMAM*7F20 and PAMAM*7F23 to promote the entry of dsRNA molecules into lepidopteran cells, the three vectors and dsRNA were further used to silence the gene expressed in the lepidopteran insect Spodoptera frugiperda Sf9 cells, and the green fluorescent protein gene (Green Fluorescent Protein, GFP) that is easy to detect the efficiency of gene silencing was selected as the target gene. The degree of decrease in GFP fluorescence intensity was used to quantitatively detect the RNAi efficiency. After the dsRNA alone interfered with GFP, the green fluorescence did not decrease within 24 hours ( Figure 2 ), the green fluorescence did not decrease significantly after the treatment time was extended to 72h, indicating that the GFP gene in Sf9 cells was expressed normally, and the RNAi efficiency of the target gene was low when the dsRNA molecules were treated in Sf9 pest cells alone. After using PAMAM, PAMAM*7F20 and PAMAM*7F23 to carry dsRNA, the green fluorescent protein was found to decrease significantly after 24h, indicating that the three vectors can greatly promote the silencing effect of dsRNA on the target gene GFP. The green fluorescence intensity was still effectively reduced to a low level after the treatment time was extended to 72h, indicating that after the three vectors carrying dsRNA were used to treat Sf9 cells, the silencing efficiency of the target gene was high and the duration was long ( Figure 2 A). The GFP fluorescence intensity at 0h, 24h, 48h and 72h after cell treatment was used to quantify the silencing efficiency of the target gene GFP. The degree of GFP fluorescence reduction caused by interfering with the GFP gene by different nanocarriers carrying dsRNA ( Figure 2 B): The GFP fluorescence of the cells in the control group treated with dsRNA alone was strong, with almost no downregulation. In contrast, the GFP fluorescence of the cells in the treatment group with the addition of the three nanocarriers was downregulated to varying degrees, among which the GFP fluorescence of the cells in the PAMAM*F23*dsRNA group was downregulated the most, indicating that the three nanocarriers can not only deliver nucleic acid drugs dsRNA into insect cells efficiently ( Figure 1 ), and can effectively improve the silencing efficiency of target genes, and the PAMAM*7F23 nanocarrier has the best improvement effect ( Figure 2-3 ).

[0029] On the basis of finding that the PAMAM*7F23 synthesized in Study Example 1 can effectively improve the efficiency of dsRNA entering cells and the efficiency of RNAi at the cellular level, we further investigated whether PAMAM*7F23 can enhance the gene interference efficiency of dsRNA and the level of pest control at the in vivo level. The larvae of the main rice pest of Lepidoptera, Chilo suppressalis, were selected as test insects, and the larvae were treated with PAMAM*7F23 / dsRNA complexes by surface dripping. After 24 hours, the larvae were washed three times with 70% ethanol and distilled water respectively to remove the PAMAM*7F23 / dsRNA complexes remaining on the insect body surface. The dsRNA red fluorescence signal can be detected in the larvae after treatment with the PAMAM*7F23 / dsRNA complex ( Figure 3 ,B-B'), while no dsRNA fluorescence signal was detected in larvae treated with dsRNA alone ( Figure 3 ,A-A'); indicating that PAMAM*7F23 significantly enhances the entry of dsRNA into living pests. The effects of PAMAM*7F23 vectors on dsRNA silencing target genes and pest control at the in vivo level were further studied. The cuticle protein gene CsLCP16 / 17, a key gene for the growth and development of Chilo suppressalis larvae, was selected as the target gene to synthesize dsRNA pest control molecules, and the PAMAM*7F23 / dsRNA complex was used to treat the body surface of the pests. The control groups were: the body surface of the pests was treated with only distilled water, the body surface of the pests was treated with only dsRNA molecules that inhibited the growth and development of the pests, the body surface of the pests was treated with only dsEGFP molecules that were harmless to the growth and development of the pests, the body surface of the pests was treated with only PAMAM*7F23 nanomaterials, and the body surface was treated with PAMAM*7F23\dsEGFP complex. The treatment group was treated with the PAMAM*7F23\dsRNA complex. In the control group, all the larvae of the stem borer bored into the rice to feed. More than 20% of the pests in the control group bored into the rice, causing the rice stems to become hollow or even broken ( Figure 3 ,CI). In contrast, after the PAMAM*7F23 / dsRNA complex was used to treat the rice stem borer, the pests all lost their rice drilling behavior, and the rice stems were intact ( Figure 3 , C and J). The PAMAM*7F23\dsRNA complex designed by the present invention 100% inhibits the damage of pests to rice at the living body level, which is helpful for the green prevention and control of the Chilo suppressalis and ensures the safe production of the food crop rice.

[0030] Although the present invention has been described in detail with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A gene transfection vector based on dendritic polyamidoamine, characterized in that: The carrier has the following structure: A-(NHCH2CH2SO2NHCH2CF2CF2CF3) n , the A is obtained by reacting n terminal amino groups of a dendritic polyamidoamine PAMAM with n vinylsulfonyl fluorides respectively and then with n terminal amino fluorides, the total terminal amino group of the dendritic polyamidoamine PAMAM is m, m is 32 or 64, and n is a natural number between 16 and m.

2. A method for preparing a gene transfection vector based on dendritic polyamidoamine, characterized in that: The method comprises the following steps: 1) a dendritic polyamidoamine PAMAM and vinylsulfonyl fluoride are mixed in a molar ratio of (1:1) to (1:40), and reacted in an organic solvent at 45-55° C. to synthesize a sulfonyl fluoride-containing dendritic polymer; 2) synthesizing fluorinated dendritic polyamidoamine by sulfonyl fluoride click polymerization reaction of sulfonyl fluoride-containing dendritic polymer and terminal amino fluoride in the presence of a catalyst at 30-50°C.

3. The method for preparing a gene transfection vector based on dendritic polyamidoamine according to claim 2, characterized in that: The organic solvent is a mixed solution of methanol and water, ethanol or DMF.

4. The method for preparing a gene transfection vector based on dendritic polyamidoamine according to claim 2 or 3, characterized in that: The amino-terminated fluoride is heptafluorobutylamine.

5. The method for preparing a gene transfection vector based on dendritic polyamidoamine according to claim 2 or 3, characterized in that: The catalyst is 1,8-diazacycloundec-7-ene or 1,5,7-triazabicyclo[4.4.0]dec-5-ene.

6. Use of the gene transfection vector based on dendritic polyamidoamine according to claim 1 as a nucleic acid dsRNA vector.

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