Polyphenol modified vector, gene delivery system and use thereof in preparing gene drug

Abstract

A polyphenol modified vector, a gene delivery system, and the use thereof in preparing a gene drug. The polyphenol modified vector comprises a vector and a polyphenol modifying same. The gene delivery system comprises the polyphenol modified vector and a gene loaded on the polyphenol modified vector.

Description

A polyphenol-modified vector, gene delivery system, and its application in preparing gene medicine Technical Field

[0001] The present invention belongs to the technical field of medical treatment and biomedical materials, and particularly relates to a polyphenol-modified carrier, a gene delivery system and applications thereof in the preparation of gene medicines. Background Art

[0002] Gene therapy is an intracellular approach that introduces exogenous therapeutic genes (transgenes) into specific host cells, eliciting therapeutic effects on genetic diseases by correcting mutations / altering genes or providing new cellular functions. It offers promising therapeutic potential and specificity for a range of diseases, including cardiovascular disease, cancer, neurological disorders, infectious diseases, and wound healing. However, naked DNA and RNA molecules are easily degraded by intracellular lysosomes or extracellular nucleases, necessitating efficient gene delivery vectors. Currently, conventional gene delivery systems require the use of large quantities of ionizable / cationic carriers to form complexes with electronegative genes (DNA, siRNA, and mRNA) through electrostatic interactions to achieve gene protection and delivery. However, conventional ionizable / cationic delivery vectors present challenges such as high toxicity, increased in vivo inflammatory responses, and cellular exocytosis.

[0003] Existing mRNA delivery mainly relies on liposome nanoparticles, but still has the following disadvantages: 1. The preparation process is cumbersome and requires the use of microfluidics, which results in a high amount of mRNA required for a single preparation; 2. Citric acid buffer solution and ethanol are used in the preparation process, which need to be removed by dialysis later. The dialysis process has high environmental requirements and can also cause mRNA degradation; 3. The delivery system cannot be degraded and is toxic in vivo; 4. The endosomal escape efficiency of the delivery system is low; 5. The delivery system is prone to cause inflammatory responses in the body.

[0004] Polyphenols possess numerous bioactivities, including antioxidant, anticancer, antibacterial, anti-inflammatory, antiviral, and cardioprotective properties, and have been widely used in various fields. Studies have shown that natural polyphenols can interact with various materials through hydrogen bonding, hydrophobic interactions, π interactions, metal coordination, electrostatic interactions, and covalent bonds. Furthermore, polyphenols possess dense ortho- and meta-phenolic hydroxyl groups that can bind to genes, making them promising candidates for gene delivery. To date, there have been no reports on the use of polyphenols as gene delivery vehicles. Summary of the Invention

[0005] In response to the above-mentioned defects in the prior art, the present invention aims to provide a polyphenol-modified vector, a gene delivery system, and its use in the preparation of gene medicines. The present invention utilizes different chemical methods to connect polyphenols to carriers to obtain polyphenol-modified vectors, which can bind to genes through hydrogen bonding or hydrophobic interactions and are further used for gene delivery. The process flow for gene loading of the polyphenol-modified vector provided by the present invention is simple and can be achieved by co-incubation at room temperature, avoiding the problems of complex preparation processes in the prior art. At the same time, the delivery system involved in the present invention is degradable and has good biosafety.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a polyphenol-modified carrier, comprising a carrier and polyphenols modified thereon.

[0008] The polyphenol-modified carrier is selected from one of polysaccharides, cationic polymers, carbon chains, metal ions or nanoparticles.

[0009] The polyphenol modified carrier is selected from one or more of flavonoids, stilbenes, phenolic acids or lignins.

[0010] The polyphenol-modified carrier is one or more of the following compounds:

[0011] The method for preparing a polyphenol-modified carrier comprises modifying the polyphenol on the carrier by using a click chemistry reaction; or modifying the polyphenol by a chemical reaction between the active group of the polyphenol and the carrier; or modifying the polyphenol by a chemical reaction between the active group of the polyphenol and the carrier modified with the active group.

[0012] In addition to click chemistry, the present invention also allows modification through reactions between polyphenols and active groups on carriers. Certain carriers lacking active groups can be modified with active groups through chemical reactions before reacting with polyphenols. These active groups include carboxyl groups, amino groups, and the like. Reactions between these active groups include amidation reactions.

[0013] A gene delivery system comprises the polyphenol modified vector and a gene carried on the polyphenol modified vector.

[0014] In the gene delivery system, the gene is selected from one of DNA, pDNA, RNA, miRNA, mRNA or siRNA.

[0015] The preparation method of the gene delivery system comprises dissolving the gene and the polyphenol-modified carrier in a solvent, and co-incubating the gene to be loaded onto the polyphenol-modified carrier through hydrogen bonding or hydrophobic interaction.

[0016] Use of any one of the polyphenol-modified vectors and the gene delivery system in the preparation of gene medicines.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a polyphenol-modified vector and a gene delivery system. The natural polyphenols used in the present invention are widely available and have good biocompatibility, antioxidant and antibacterial properties. Natural polyphenols have a compact polyphenol structure and can bind to genes through hydrogen bonding or hydrophobic interactions. The preparation method of the present invention is simple, and the raw materials used are environmentally friendly, low-cost, easy to operate, and low-cost. Experimental results have shown that the polyphenol-modified vector can effectively deliver genes and can demonstrate good gene delivery efficiency. The polyphenol-modified vector can deliver genes and improve their transfection efficiency, and has good application prospects in gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 is a schematic diagram of a polyphenol-modified gene-loaded vector prepared in Example 1 of the present invention;

[0020] FIG2 is the structural formula of the polyphenol-modified carrier prepared in Example 1 of the present invention;

[0021] FIG3 is a gel electrophoresis pattern of the gene-loaded polyphenol-modified vector prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0022] In order to better understand the present invention, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but the content of the present invention is not limited to the following embodiments.

[0023] Example 1:

[0024] 1) Preparation method of quercetin modified carrier: First, polyphenols are modified to octadecyl mercaptan by click chemistry reaction to obtain a polyphenol-modified carrier. The specific process is: quercetin is dissolved in ethanol, ultrasonically dispersed, and then the pH is adjusted to 7-8 with 1M sodium hydroxide, stirred at room temperature, and then octadecyl mercaptan is dissolved in ethanol and added dropwise to the above system; finally, the reaction is carried out under ultraviolet irradiation of 365nm for 3-5 hours. After the reaction is completed, the quercetin-modified 18-carbon chain obtained by the reaction is purified for 2-3 days using a dialysis tube (300Da), and then freeze-dried and stored at 4°C for further use. Figure 2 is the structural formula of the polyphenol-modified carrier and each monomer synthesized in this embodiment, wherein A is the structural formula of quercetin, B is the structural formula of octadecyl mercaptan, and C is the structural formula of quercetin-modified octadecyl mercaptan.

[0025] 2) Gene-carrying method: The quercetin-modified 18-carbon chain was dissolved in dimethyl sulfoxide, and then lipid nanoparticles were prepared with polyethylene glycol, cholesterol and mRNA gene through microfluidic technology to successfully carry the gene.

[0026] The specific experimental steps for testing gene binding capacity using agarose gel are as follows: First, prepare a 1% agarose gel and add Gel Red to fully blend it with the agarose. Second, pour the agar into the prepared gel mold, insert a comb, and ensure that there are no bubbles. Wait for it to solidify. After solidification, remove the comb and place it in the electrophoresis tank. Next, mix the sample to be analyzed with the loading buffer in a specified ratio and add it to the sample tank. Finally, close the electrophoresis tank lid and connect the power supply to 110V. Run the electrophoresis for approximately 20-30 minutes, then stop the electrophoresis and take photos and observe.

[0027] Figure 3 shows the gel electrophoresis pattern of the polyphenol-modified vector synthesized in this example bound to the gene. As can be seen from the figure, naked mRNA is gradually released during electrophoresis, while mRNA bound to the polyphenol-modified vector is not released, indicating that the polyphenol-modified vector has good gene binding ability.

[0028] Example 2:

[0029] 1) Preparation method of tannic acid modified carrier: First, tannic acid is modified to octadecyl mercaptan through a click chemistry reaction to obtain a polyphenol-modified carrier. The specific process is: tannic acid is dissolved in ethanol, ultrasonically dispersed, and then the pH is adjusted to 7-8 with 1M sodium hydroxide, stirred at room temperature, and then octadecyl mercaptan is dissolved in ethanol and added dropwise to the above system; finally, the reaction is carried out under ultraviolet irradiation at 365nm for 3-5 hours. After the reaction is completed, the tannic acid-modified octadecyl chain obtained by the reaction is purified for 2-3 days using a dialysis tube (300Da), then freeze-dried and stored at 4°C for further use.

[0030] 2) Gene loading method: The tannic acid-modified octadecyl chain was dissolved in dimethyl sulfoxide, and then prepared with polyethylene glycol, cholesterol and genes through microfluidic technology to prepare lipid nanoparticles, which successfully loaded the genes.

[0031] Example 3:

[0032] 1) Preparation of epigallocatechin gallate (EGCG) modified carrier: First, EGCG is modified to octadecyl mercaptan through a click chemistry reaction to obtain a polyphenol-modified carrier. The specific process is as follows: EGCG is dissolved in ethanol and ultrasonically dispersed, then the pH is adjusted to 7-8 with 1M sodium hydroxide and stirred at room temperature. Subsequently, octadecyl mercaptan is dissolved in ethanol and added dropwise to the above system; finally, the reaction is carried out under ultraviolet irradiation at 365nm for 3-5 hours. After the reaction is completed, the EGCG-modified octadecyl chain obtained by the reaction is purified for 2-3 days using a dialysis tube (300Da), then freeze-dried and stored at 4°C for further use.

[0033] 2) Gene loading method: The 18-carbon chain modified with EGCG was dissolved in dimethyl sulfoxide, and then lipid nanoparticles were prepared with polyethylene glycol, cholesterol and genes through microfluidic technology to successfully load the genes.

[0034] Example 4:

[0035] 1) Preparation of Gallic Acid-Modified Carriers: Gallic acid is modified onto amino-containing polysaccharides using an amidation reaction. Gallic acid is first dissolved in MES buffer (pH = 5-6) and stirred to dissolve. Activated with NHS and EDC, after 10 hours, the amino-containing polysaccharide is added and stirred for 2-3 days. Finally, the gallic acid-modified carrier obtained by the reaction is purified using a dialysis tube (10 kDa) for 2-3 days. It is freeze-dried and stored at 4°C for further use.

[0036] 2) Gene loading method: Dissolve gallic acid-modified polysaccharide in DEPC water and incubate with the gene for 30 minutes to bind the gene through hydrogen bonding or hydrophobic interaction.

[0037] Example 5:

[0038] 1) Preparation of Dopamine-Modified Carrier: Dopamine was modified onto hyaluronic acid using an amidation reaction. Dopamine was first dissolved in MES buffer (pH 5-6) with stirring. Activation was then performed with NHS and EDC. After 10 hours, hyaluronic acid was added and stirring continued for 2-3 days. Finally, the resulting dopamine-modified carrier was purified using dialysis tubing (10 kDa) for 2-3 days. The product was freeze-dried and stored at 4°C for further use.

[0039] 2) Gene loading method: dissolve dopamine-modified hyaluronic acid in DEPC water and incubate with the gene for 30 minutes to bind to the gene through hydrogen bonding or hydrophobic interaction.

[0040] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A polyphenol-modified carrier, characterized in that, It contains a carrier and polyphenols modified thereon.

2. The polyphenol-modified carrier according to claim 1, wherein, The carrier is selected from one of polysaccharides, cationic polymers, carbon chains, metal ions or nanoparticles.

3. The polyphenol-modified carrier according to claim 1, characterized in that, The polyphenols are selected from one or more of flavonoids, stilbenes, phenolic acids or lignins.

4. A polyphenol-modified carrier according to claim 1, wherein The polyphenol is one or more of the compounds with the following structural formulas:

5. The preparation method of a polyphenol-modified carrier according to claim 1, characterized in that, The polyphenols are modified on the carrier by click chemistry reaction; or, modified by the chemical reaction between the active groups of the polyphenols and the carrier; or, modified by the chemical reaction between the active groups of the polyphenols and the carrier modified with active groups.

6. A gene delivery system, characterized in that, It contains the polyphenol-modified carrier described in claim 1 and a gene carried on the polyphenol-modified carrier.

7. A gene delivery system according to claim 6, wherein The gene is selected from one of DNA, pDNA, RNA, miRNA, mRNA or siRNA.

8. The preparation method of a gene delivery system according to claim 6 or 7, characterized in that, The gene and the polyphenol-modified carrier are dissolved in a solvent and co-incubated to carry the gene onto the polyphenol-modified carrier through hydrogen bond action or hydrophobic interaction.

9. Use of the polyphenol-modified carrier according to any one of claims 1-4 and the gene delivery system according to claim 6 or 7 in the preparation of gene drugs.

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