Hyperbranched polymer dsRNA delivery carrier as well as preparation method and application thereof

Abstract

The invention discloses a hyperbranched polymer dsRNA (double-stranded ribonucleic acid) delivery carrier. The vector 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, and the prepared vector is safe, low in toxicity and excellent in water solubility, has efficient gene transfection efficiency and low toxicity, and has a relatively high application prospect in the field of agricultural pest control.

Description

Technical Field

[0001] The invention belongs to the technical fields of polymer chemistry, biomaterials and agricultural pest control, and specifically relates to a hyperbranched polymer dsRNA delivery carrier 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. With the development of advanced biotechnology therapies, 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 pest control and plant protection. 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] Currently, commonly used nucleic acid vectors mainly include viral and non-viral types. Viral gene transfection vectors have safety issues and the limitations of low gene transfection efficiency, which limit their scope of application. The cationic nucleic acid vectors that have been studied more are polyethyleneimine, polylysine and polyamide-amine hyperbranched polymers, among which the application research of hyperbranched polymers is particularly valued. There are a large number of cavities inside the hyperbranched polymer molecules and they carry a large number of surface functional groups. They can be widely used in the field of biomedicine, especially polyamide-amine hyperbranched polymers, which have been widely studied and applied in gene transfection due to their high efficiency, safety and low toxicity. However, due to the differences in the types of organisms, the transfection effect and biocompatibility of the same hyperbranched polymer carrier on different organisms may be different. In order to effectively protect genetic material and promote the cellular uptake and cell endosome escape of gene complexes, ensure the efficient expression of genetic material, and effectively prevent and control agricultural pests, it is necessary to further screen and modify the vector in a targeted manner. Summary of the invention

[0005] The first technical problem to be solved by the present invention is to provide a hyperbranched polymer dsRNA delivery vector. The vector has good water solubility and biocompatibility, can enter living cells, and can bind to negatively charged nucleic acid molecules dsRNA through electrostatic forces, so that it can be used as a nucleic acid vector to carry exogenous nucleic acid component dsRNA into cells.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing the hyperbranched polymer dsRNA delivery vector.

[0007] In order to solve the above-mentioned first technical problem, the hyperbranched polymer dsRNA delivery vector provided by the present invention has the following structure:

[0008]

[0009] The vector may have a structure of The hyperbranched polymer is obtained by reacting one terminal amino group with one vinyl sulfonyl fluoride and then with one terminal amino fluoride, wherein the structural formula of A is: The structural formula of B is The structural formula of D is: E is a group formed by the amino group of the terminal amino fluoride losing a hydrogen, each A is bonded to three Bs through its three free ends, and each B is bonded to two As through its two free ends, and the outermost A is bonded to two Hs to become a primary amine; m is a natural number of 0 to 8, n is a natural number of 8 to 16, and m+n=16; p+q=m, x+y=2n, y+q=1; wherein x, y, p and q are natural numbers.

[0010] To solve the above second technical problem, the preparation method provided by the present invention comprises the following steps:

[0011] (1) A hyperbranched polyamidoamine and vinyl sulfonyl fluoride are mixed in a molar ratio of (1:1) to (1:30), and reacted in an organic solvent at 45-50° C. to synthesize a hyperbranched polymer containing sulfonyl fluoride.

[0012] (2) The sulfonyl fluoride hyperbranched polymer and the terminal amino fluoride (same molar amount as vinyl sulfonyl fluoride) are subjected to a sulfonyl fluoride click polymerization reaction at 30-50° C. in the presence of a catalyst to synthesize a fluorinated hyperbranched polysulfonamide.

[0013] Preferably, the organic solvent is a mixed solution of methanol and water, ethanol or DMF.

[0014] Preferably, the terminal amino fluoride is heptafluorobutylamine.

[0015] Preferably, the catalyst is 1,8-diazacycloundec-7-ene (DBU) or 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD).

[0016] Preferably, said l is a natural number between 16 and 32.

[0017] The following formula, labeled as HPAA-7F18, is a hyperbranched polymer dsRNA delivery carrier structure in which m is 8, n is 8, and l is 18 (the terminal amino group is heptafluorobutylamine):

[0018]

[0019] The present invention also provides the use of the hyperbranched polymer dsRNA delivery vector as a nucleic acid vector.

[0020] Preferably, the application is used to deliver dsRNA insecticidal molecules to control fall armyworm.

[0021] The synthesized fluorinated hyperbranched polysulfonamide is incubated with living cells. The fluorinated hyperbranched polysulfonamide can quickly and efficiently enter the living cells of pests, and can be combined with exogenous nucleic acid substances dsRNA, as a carrier, carrying exogenous nucleic acid substances to efficiently and quickly transfect living cells. The fluorinated hyperbranched polysulfonamide / dsRNA complex is further mixed into insect feed and fed to pests, and it can penetrate the peritrophic membrane of the intestine and the intestinal cell barrier to enter the intestinal cells, indicating that the fluorinated hyperbranched polysulfonamide can carry dsRNA into the living larvae, that is, the fluorinated hyperbranched polysulfonamide has the conditions for being used as a nucleic acid carrier in vitro or in vivo.

[0022] The present invention has the following beneficial effects:

[0023] 1. The present invention utilizes Michael addition and sulfonyl fluoride click polymerization to carry out the reaction, and can prepare hyperbranched polysulfonamide with controllable fluorine content.

[0024] 2. The fluorinated hyperbranched polysulfonamide prepared by the method of the present invention has good biocompatibility, and the hyperbranched macromolecules of the fluorinated alkyl chain can improve the cell's uptake of exogenous nucleic acids and further promote the release of dsRNA to the target position in the cell, thereby improving the transfection efficiency of nucleic acids, and can therefore be used as a nucleic acid carrier to carry exogenous nucleic acids into cells. Experiments have shown that the synthesized fluorinated hyperbranched polysulfonamide has high gene transfection efficiency and low toxicity, and has good application value.

[0025] The invention has the advantages of simple synthesis, high efficiency, simple product purification, etc., and the synthesized fluorinated hyperbranched polysulfonamide 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 gene therapy in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The HPAA-7F series nanocarriers in Example 5 of the present invention deliver nucleic acid dsRNA to quickly enter cells.

[0027] Figure 2 The intestines of the pests in Example 6 were dissected 3 days after they had eaten dsRNA / HPAA-7F18. The dsRNA broke through the peritrophic membrane (PM) from the intestinal food and entered the intestinal cells.

[0028] Figure 3 In Example 7, after feeding the pests with HPAA-7F18@dsRNA, the expression level of the target gene of the pest Spodoptera frugiperda was effectively silenced (a), the body weight of the pests was effectively reduced (b), and the gnawing damage of the pests to the leaves of corn crops was effectively reduced (cd). DETAILED DESCRIPTION

[0029] 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 raw materials used are all commercially available products.

[0030] Example 1 Preparation of hyperbranched polyamidoamine

[0031] The hyperbranched polyamidoamine was synthesized according to the literature reported by Professor Ye-Zi You. The specific experimental steps refer to the literature (Ye-Zi You, Zhi-Qiang Yu, Meng-Meng Cui, Chun-Yan Hong, Preparation of Photoluminescent Nanorings with Controllable Bioreducibility and Stimuli-Responsiveness, Angewandte Chemie International Edition. 2010, 49(6), 1099-1102.). N,N-methylenebisacrylamide (0.3083 g, 2 mmoL) and aminopiperazine (2.584 g, 20 mmoL) were mixed in a molar ratio of 1:10. The reaction conditions were as follows: added to 6.0 mL of a mixed solvent of methanol and water, with a volume ratio of methanol to water of 4:1, and then stirred at room temperature for 24 h under a nitrogen atmosphere, and then reacted at 53 ° C for 2 days. After the reaction temperature dropped to room temperature, it was washed with a mixed solution of ether and n-hexane in a volume ratio of 4:1, and vacuum dried to obtain hyperbranched polyamidoamine (HPAA). 1H NMR(400MHz, DMSO-d6)δ8.82-8.60(m,6H),6.25(ddd,J=17.2,10.0,4.3Hz,4H),6.19-6.07(m,4H),5.66-5.5 7(m,4H),4.50-4.34(m,11H),2.54-2.42(m,23H),2.30(s,8H),2.20(dq,J=12.8,6.4Hz,22H),2.09(s,20H).

[0032] Example 2 Preparation of heptafluoro hyperbranched polysulfonamide

[0033] Hyperbranched polyamidoamine (1.350 g, 0.1 mmoL) was reacted with vinylsulfonyl fluoride (0.2643 g, 2.4 mmoL) in ethanol solvent at 50°C to synthesize a hyperbranched polymer containing sulfonyl fluoride, and then reacted with heptafluorobutylamine (0.3981 g, 2 mmoL) in the presence of TBD as a catalyst and EtOH as a solvent at 50°C to synthesize a fluorinated hyperbranched polysulfonamide (HPAA-7F20) through sulfonyl fluoride click polymerization. 1 H NMR(400MHz,DMSO-d6)δ8.64-8.56(m,28H),4.36(d,J=8.8Hz,50H),3.44(d,J =7.0Hz,58H),3.31-3.15(m,58H),3.08(t,J=5.8Hz,29H),2.82(t,J=6.4Hz,47 H),2.76-2.57(m,163H),2.47(s,121H),2.43-2.34(m,108H),2.28-2.17(m,11 0H), 1.20 (td, J=14.1, 12.2, 6.0Hz, 15H), 1.05 (t, J=7.0Hz, 75H), 0.95 (s, 5H). 19 F NMR(377MHz,D2O)δ:-80.76(s,3F),-118.89(s,2F),-128.19(s,2F).

[0034] Hyperbranched polyamidoamine (1.350 g, 0.1 mmoL) was reacted with vinylsulfonyl fluoride (0.1376 g, 1.25 mmoL) in DMF solvent at 50°C to synthesize a hyperbranched polymer containing sulfonyl fluoride, and then reacted with heptafluorobutylamine (0.2389 g, 1.2 mmoL) in the presence of TBD as a catalyst and EtOH as a solvent at 50°C to synthesize a fluorinated hyperbranched polysulfonamide (HPAA-7F12) through sulfonyl fluoride click polymerization.

[0035] Hyperbranched polyamidoamine (1.350 g, 0.1 mmoL) was reacted with vinyl sulfonyl fluoride (0.2037 g, 1.85 mmoL) in ethanol solvent at 50°C to synthesize a sulfonyl fluoride-containing hyperbranched polymer, and then reacted with heptafluorobutylamine (0.3583 g, 1.8 mmoL) in the presence of TBD as a catalyst and EtOH as a solvent at 50°C to synthesize a fluorinated hyperbranched polysulfonamide (HPAA-7F18) through sulfonyl fluoride click polymerization.

[0036] Example 3 Preparation of Pentafluoro Hyperbranched Polysulfonamide

[0037] Hyperbranched polyamidoamine (1.350 g, 0.1 mmoL) was reacted with vinylsulfonyl fluoride (0.2643 g, 2.4 mmoL) in ethanol solvent at 50°C to synthesize a hyperbranched polymer containing sulfonyl fluoride, and then reacted with pentafluoropropylamine (0.3261 g, 2 mmoL) in DBU as a catalyst and DMF as a solvent at 30°C to synthesize a fluorinated hyperbranched polysulfonamide (HPAA-5F).

[0038] Example 4 Preparation of hyperbranched polysulfonamide containing pentafluoroaniline

[0039] Hyperbranched polyamidoamine (1.350 g, 0.1 mmoL) was reacted with vinylsulfonyl fluoride (0.2643 g, 2.4 mmoL) in DMF solvent at 50°C to synthesize a hyperbranched polymer containing sulfonyl fluoride. Finally, it was reacted with pentafluoroaniline (0.3261 g, 2 mmoL) in the presence of DBU as a catalyst and DMF as a solvent at 40°C to synthesize a fluorinated hyperbranched polysulfonamide (HPAA-5F) through sulfonyl fluoride click polymerization.

[0040] Example 5 Living cell uptake experiment

[0041] The nanocarriers HPAA, HPAA-7F12, and HPAA-7F18 in Example 2 were assembled with dsRNA nucleic acid drugs to form nanocarrier / dsRNA complex drugs. A dsRNA efficient delivery system was established for the lepidopteran model Spodoptera frugiperda Sf9 cells, which can quickly deliver trace amounts of dsRNA (0.125 μg / mL) into Sf9 cells (e.g. Figure 1 24h detection of dsRNA fluorescence intensity in Sf9 cells revealed that HPAA, HPAA-7F12, and HPAA-7F18 could promote dsRNA to break through the cell membrane barrier of Sf9 cells and improve the efficiency of dsRNA entering cells. It was found that the efficiency of carrying dsRNA into cells was HPAA-7F18 / dsRNA>HPAA-7F12 / dsRNA>HPAA / dsRNA (as shown in Figure 1The nano-delivery system based on HPAA-7F18 breaks through the inherent bottleneck of degrading dsRNA drugs in Lepidoptera cells, and provides technical support for the further development of efficient RNAi systems.

[0042] The effects of the pentafluoro-containing hyperbranched polysulfonamide and the pentafluoroaniline-containing hyperbranched polysulfonamide prepared in Examples 3 and 4 are significantly lower than those of HPAA-7F12 and HPAA-7F18.

[0043] Example 6: Vector carrying dsRNA into living pests

[0044] In order to study the transfection efficiency of HPAA-7F18 synthesized in Example 2 at the in vivo level, it was investigated whether the HPAA-7F18 / dsRNA complex can enter the larvae. The HPAA-7F18 / dsRNA complex was mixed into insect feed and fed to pests. HPAA-7F18 / dsRNA can penetrate the intestinal peritrophic membrane and the intestinal cell barrier and enter the intestinal cells (such as Figure 2 ), indicating that HPAA-7F18 can carry dsRNA into living larvae. After the living pests ingested dsRNA / H18 for 7 days, dsRNA spread from the intestine to the body. These experimental results show that HPAA-7F18 has the conditions for use as a nucleic acid carrier in vitro or in vivo.

[0045] Example 7: Vector-carrying dsRNA for controlling the pest Spodoptera frugiperda

[0046] The fall armyworm, a major agricultural pest in the world, was taken as the research object, and the chitin synthase gene CHS2, a key gene for controlling the growth and development of the fall armyworm, was taken as the target gene. The HPAA-7F18 vector synthesized in Example 2 was used to establish a vector system for delivering dsRNA. The HPAA-7F18@dsRNA complex was mixed with feed and fed to the fall armyworm larvae in the early third instar. Compared with the control group (fed only with dsRNA insecticidal molecules, fed only with HPAA-7F18 nanocarriers, fed only with dsEGFP, and fed with HPAA-7F18@dsEGFP), the feeding of the HPAA-7F18@dsRNA system effectively reduced various pest indicators ( Figure 3 , ad), compared with feeding dsRNA molecules alone, the inhibition rate of the expression level of the key gene chitin synthase gene of the pest was increased by 79.8% ( Figure 3 a), the inhibition rate of pest weight increased by 54.5% ( Figure 3 b), effectively reducing the damage to corn leaves by 69.3% ( Figure 3 , cd).

[0047] 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 hyperbranched polymer dsRNA delivery vector, characterized in that: The delivery vector has the following structure: The vector may have a structure of The hyperbranched polymer is obtained by reacting one terminal amino group with one vinyl sulfonyl fluoride and then with one terminal amino fluoride, wherein the structural formula of A is: The structural formula of B is The structural formula of D is: E is a group formed by the amino group of the terminal amino fluoride losing a hydrogen, each A is bonded to three Bs through its three free ends, and each B is bonded to two As through its two free ends, and the outermost A is bonded to two Hs to become a primary amine; m is a natural number of 0 to 8, n is a natural number of 8 to 16, and m+n=16; p+q=m, x+y=2n, y+q=1, wherein x, y, p and q are natural numbers.

2. A method for preparing a hyperbranched polymer dsRNA delivery vector, characterized in that: The method comprises the following steps: (1) mixing a hyperbranched polyamidoamine and vinyl sulfonyl fluoride in a molar ratio of (1:1) to (1:30), and reacting them in an organic solvent at 45 to 50° C. to synthesize a hyperbranched polymer containing sulfonyl fluoride; (2) A sulfonyl fluoride hyperbranched polymer is reacted with an amino-terminated fluoride in the presence of a catalyst at 30-50° C. to synthesize a fluorinated hyperbranched polysulfonamide.

3. The method for preparing the hyperbranched polymer dsRNA delivery vector 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 the hyperbranched polymer dsRNA delivery vector according to claim 2 or 3, characterized in that: The terminal amino fluoride is heptafluorobutylamine.

5. The method for preparing the hyperbranched polymer dsRNA delivery vector 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. The method for preparing the hyperbranched polymer dsRNA delivery vector according to claim 2 or 3, characterized in that: The sum of the natural numbers y and q is a natural number between 16 and 32.

7. Use of the hyperbranched polymer dsRNA delivery vector according to claim 1 as a nucleic acid dsRNA vector.

8. The use according to claim 7, characterized in that: The application as a nucleic acid dsRNA carrier refers to delivering dsRNA insecticidal molecules to control fall armyworm.

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