阅读说明：本技术 一种多功能核酸基杂化纳米凝胶及其制备方法 (Multifunctional nucleic acid-based hybrid nanogel and preparation method thereof ) 是由 仰大勇 韩金鹏 崔宇辰 于 2019-09-06 设计创作，主要内容包括：本发明公开了一种多功能核酸基杂化纳米凝胶及其制备方法,步骤为：(1)含粘性末端的多支状核酸纳米结构的制备；(2)将步骤(1)所得的具有互补序列粘性末端的多支状核酸纳米结构进行等量混合,制备树枝状核酸网络纳米结构；(3)将步骤(2)得到的树枝状核酸网络纳米结构和多酚羟基的小分子或高分子化合物进行混合,制备多功能核酸基杂化纳米凝胶。本发明的核酸基杂化纳米凝胶,制备过程简单,方法绿色环保,通过改变树枝状核酸网络纳米结构和多酚羟基的小分子或高分子化合物的比例可精准调控纳米凝胶尺寸,实现高效的细胞摄取效率。通过充分利用核酸纳米结构的多功能性和可编程性,进行高效的基因/化学联合治疗。(The invention discloses a multifunctional nucleic acid-based hybrid nanogel and a preparation method thereof, and the preparation method comprises the following steps: (1) preparing a multi-branched nucleic acid nanostructure containing sticky ends; (2) equivalently mixing the multi-branched nucleic acid nano-structures with complementary sequence adhesive tail ends obtained in the step (1) to prepare a dendritic nucleic acid network nano-structure; (3) and (3) mixing the dendritic nucleic acid network nano structure obtained in the step (2) with micromolecules or high molecular compounds of polyphenol hydroxyl to prepare the multifunctional nucleic acid-based hybrid nano gel. The nucleic acid-based hybrid nanogel disclosed by the invention is simple in preparation process, the method is green and environment-friendly, the size of the nanogel can be accurately regulated and controlled by changing the ratio of the dendritic nucleic acid network nanostructure to the micromolecule or high-molecular compound of polyphenol hydroxyl, and the efficient cell uptake efficiency is realized. By fully utilizing the multifunctionality and programmability of nucleic acid nanostructures, efficient gene/chemical combination therapy is performed.)

1. The preparation method of the multifunctional nucleic acid-based hybrid nanogel is characterized by comprising the following steps of:

(1) mixing a plurality of single-stranded nucleic acid sequences with equal molar ratio in equal proportion, adding a cation buffer solution, performing Polymerase Chain Reaction (PCR), and preparing a multi-branched nucleic acid nanostructure containing single-stranded sticky ends by the base complementary pairing principle;

(2) equivalently mixing the multi-branched nucleic acid nano structure with the complementary nucleic acid sequence single-chain sticky tail end obtained in the step (1), wherein the mixing temperature is 4-40 ℃, the vibration rotating speed is 0-3000 rpm, and the reaction time is 0.5 h-3 d, so as to prepare the dendritic nucleic acid network nano structure;

(3) and (3) mixing the dendritic nucleic acid network nano structure obtained in the step (2) with micromolecules or high molecular compounds of polyphenol hydroxyl according to a ratio, standing at room temperature, and preparing the multifunctional nucleic acid-based hybrid nanogel.

2. The method for preparing the multifunctional nucleic acid-based hybrid nanogel according to claim 1, wherein the single-stranded nucleic acid sequence in the step (1) has a base sequence complementary to other single strands and a viscous terminal base sequence which is not complementary, and the ratio of the lengths of the two parts of the base sequence to the base sequence is 1: 1-5: 1.

3. The method for preparing the multifunctional nucleic acid-based hybrid nanogel according to claim 1, wherein the number of the plurality of single-stranded nucleic acid sequences in the step (1) is 2-5.

4. The method for preparing the multifunctional nucleic acid-based hybrid nanogel according to claim 1, wherein the single-stranded nucleic acid sequence in the step (1) is deoxyribonucleic acid or ribonucleic acid.

5. The method for preparing multifunctional nucleic acid-based hybrid nanogel according to claim 1, wherein the cation in the cation buffer solution in step (1) is Na⁺，Mg²⁺、Ca²⁺、Zn²⁺Or Fe²⁺Any one to more than one of mixed; the buffer solution is any one or more of phosphate buffer solution, acetate buffer solution, TAE buffer solution and the like.

6. The method of claim 1, wherein the multi-functional nucleic acid-based hybrid nanogel is formed by mixing the number of complementary sticky ends of the multi-branched nucleic acid nanostructure in step (2) in equal amounts, so as to ensure the formation of a uniform and complete dendritic nucleic acid network nanostructure.

7. The method for preparing the multifunctional nucleic acid-based hybrid nanogel as claimed in claim 1, wherein the small molecule or high molecular compound containing the phenolic hydroxyl group of the polyphenol in the step (3) is a compound containing the phenolic hydroxyl group on the benzene ring, and comprises gallic acid, pyrogallic acid, endorphin, tannic acid, epigallocatechin gallate, tea polyphenol, pyrogallol, dopamine, 3, 4, 5-trihydroxy phenylalanine or catechol or a high molecular compound modified by a triphenol group.

8. The method of claim 7, wherein the catechol or triphenol group-modified polymer compound comprises polyethylene glycol, dextran, cellulose, methyl cellulose, hyaluronic acid, chitosan, polylactic acid, polyvinyl alcohol, or polyvinylpyrrolidone.

9. The method for preparing multifunctional nucleic acid-based hybrid nanogel according to claim 1, wherein the dendritic nucleic acid network nanostructure and the small molecule or polymer compound of the polyphenol hydroxyl group in the step (3) are mixed in proportion, the concentration of the solution of the dendritic nucleic acid network nanostructure is 0.1 to 50 μ M, the mass concentration of the solution of the small molecule or polymer compound of the polyphenol hydroxyl group is 0.1 to 50 w/v%, and the volume ratio of the mixture of the two is (1 to 100): (1 to 100).

10. The multifunctional nucleic acid-based hybrid nanogel prepared by the method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the field of nucleic acid-based nano biomedicine, and particularly relates to a multifunctional nucleic acid-based hybrid nanogel and a preparation method thereof.

Background

Since the end of the 20 th century, the vigorous development of nano-fabrication and biotechnology has made the research of nano-materials more and more important, and a series of related research fields and industrial chains have been promoted, playing more and more important roles in the fields of environment, energy, biomedicine, and the like. The characteristic size of the nano material is between 1 and 100nm, thereby endowing the nano material with some special physical and chemical characteristics different from the conventional size material, such as remarkable surface and interface effects, small size effects, quantum size effects, macroscopic quantum tunneling effects and the like. These special properties make it show many advantages in practical applications, such as large specific surface area, high reactivity, strong adsorption capacity, strong catalytic capacity, and low toxicity. With the further development of nano science, nano materials successfully occupy a significant position in the fields of energy, information, fine chemical engineering, biomedicine and the like by virtue of excellent properties of the nano materials. Among them, biomedical nanomaterials are becoming a focus of recent research, and are used as anticancer therapeutic agents, drug delivery carriers, and the like.

Currently, delivery vectors mainly include two major types, viral vectors and non-viral vectors. Viral vectors have safety issues, especially immunogenicity and mutagenic toxicity, and are generally considered to be high risk. The non-viral vector mainly comprises liposome, polymer nanoparticles, micelle, inorganic nanoparticles and the like. However, these vectors also have drawbacks such as low biocompatibility, structural instability, difficulty in degradation in vivo, etc., and are also limited by transfection efficiency and systemic toxicity changes after injection.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a multifunctional nucleic acid-based hybrid nanogel and a preparation method thereof, and solves the problems of low transfection efficiency, poor degradability, structural instability and systemic toxicity of the existing nanocarrier.

The technical scheme of the invention is summarized as follows:

a preparation method of multifunctional nucleic acid-based hybrid nanogel mainly comprises three steps, namely:

(1) mixing a plurality of single-stranded nucleic acid sequences with equal molar ratio in equal proportion, adding a cation buffer solution, performing Polymerase Chain Reaction (PCR), and preparing a multi-branched nucleic acid nanostructure containing single-stranded sticky ends by the base complementary pairing principle;

(2) equivalently mixing the multi-branched nucleic acid nano structure with the complementary nucleic acid sequence adhesive tail end obtained in the step (1), wherein the mixing temperature is 4-40 ℃, the vibration rotating speed is 0-3000 rpm, and the reaction time is 0.5 h-3 d, so as to prepare a dendritic nucleic acid network nano structure;

(3) and (3) mixing the dendritic nucleic acid network nano structure obtained in the step (2) and the micromolecule or the macromolecular compound of the polyphenol hydroxyl according to a proper proportion, standing at room temperature, and preparing the multifunctional nucleic acid-based hybrid nano gel.

The single-stranded nucleic acid sequence has a base sequence complementary with other single strands and also has an uncompensated sticky terminal base sequence, the length ratio of the two parts of the sequence is 1: 1-5: 1, the number of the nucleic acid sequences is 2-5, and the nucleic acid sequence can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

The cation is Na⁺，Mg²⁺、Ca²⁺、Zn²⁺Or Fe²⁺Etc., preferably Mg²⁺(ii) a The buffer solution is any one or mixture of at least two of phosphate buffer solution, acetate buffer solution, TAE buffer solution and the like, and preferably TAE buffer solution.

The equal mixing of the multi-branched nucleic acid nanostructures means that the number of complementary cohesive ends of the multi-branched nucleic acid nanostructures is equal, ensuring that a uniform and complete dendritic nucleic acid network nanostructure is formed.

The micromolecule or high molecular compound of the polyphenol hydroxyl is a compound containing phenolic hydroxyl on benzene ring, and comprises gallic acid, pyrogallic acid, endorphin, tannic acid, epigallocatechin gallate, tea polyphenol, pyrogallol, dopamine, 3, 4, 5-trihydroxy phenylalanine or catechol or high molecular compound modified by a triphenol group. Wherein the macromolecular compound modified by catechol or triphenol group comprises polyethylene glycol, dextran, cellulose, methylcellulose, hyaluronic acid, chitosan, polylactic acid, polyvinyl alcohol or polyvinylpyrrolidone.

The dendritic nucleic acid network nano structure and the micromolecules or high molecular compounds of the polyphenol hydroxyl are mixed according to a proper proportion, specifically, the concentration of the dendritic nucleic acid network nano structure solution is 0.1-50 mu M, the mass concentration of the micromolecules or high molecular compounds of the polyphenol hydroxyl is 0.1-50 w/v%, and the mixing volume ratio of the dendritic nucleic acid network nano structure solution to the polyphenol hydroxyl is (1-100): 1-100.

The multifunctional nucleic acid-based hybrid nanogel is prepared according to the method.

The invention has the beneficial effects that: the method can accurately adjust the size of the nano particles by changing different proportions of the used substances so as to form the nucleic acid-polyphenol hydroxyl compound nano material, and the prepared multifunctional nucleic acid-based hybrid nano gel can make up the limitations of other carriers, fully exerts high biocompatibility, structural designability and functional programmability, and is used for a high-efficiency gene or medicine delivery system.

The invention adopts an environment-friendly method to prepare the novel multifunctional nucleic acid-based hybrid nanogel, the preparation process is simple, the preparation method is environment-friendly, the size of the nanogel can be accurately regulated and controlled by changing the ratio of the dendritic nucleic acid network nanostructure to the micromolecule or the macromolecular compound of the polyphenol hydroxyl, and the efficient cell uptake efficiency is realized. In addition, by fully utilizing the multifunctionality and the programmability of the nucleic acid nano structure, the nanogel can simultaneously load a plurality of nucleic acid sequences with different biological effects, and meanwhile, the hydrophobicity of the nucleic acid base can load hydrophobic drugs to carry out efficient gene/chemical combination treatment, so that the nanogel has very wide application prospect in the field of nano biomedicine.

The design principle of the invention is to fully utilize the base complementary pairing and the programmability principle of nucleic acid molecules, form a novel nucleic acid nano material through the interaction of hydrogen bonds, pi-pi accumulation and the like between the nucleic acid nano material and polyphenol hydroxyl compounds or derivatives thereof, and adjust the size of nucleic acid nano particles by adjusting the concentration ratio of the polyphenol hydroxyl compounds or the derivatives thereof to the nucleic acid molecules.

Drawings

FIG. 1 is a representation of the product of example 1 at various stages of the process for the preparation of dendritic DNA network nanostructures by gel electrophoresis;

FIG. 2 is a Scanning Electron Microscope (SEM) morphology of the multifunctional DNA hybrid nanogel prepared in example 1;

FIG. 3 is a projection electron microscope (TEM) morphology image of the RNA-based hybrid nanogel prepared in example 4;

FIG. 4 is a graph of the dynamic particle size statistics (DLS) of nanogels of different sizes of DNA-RNA hybrid nanogels prepared in example 5, the size of which can be adjusted by varying the content of nucleic acid;

FIG. 5 is the drug release profile of DNA-based hybrid nanogel prepared in example 6 at different pH conditions after loading with anticancer drug Doxorubicin (DOX).

Detailed Description

The present invention will be further illustrated by the following specific examples.

The following examples are intended to enable those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.

A preparation method of multifunctional nucleic acid-based hybrid nanogel comprises the following steps:

(1) preparation of a multi-branched nucleic acid nanostructure containing single-stranded sticky ends: mixing a plurality of single-stranded nucleic acid sequences with equal molar ratio in equal proportion, adding a cation buffer solution, performing Polymerase Chain Reaction (PCR), and preparing a multi-branched nucleic acid nanostructure containing single-stranded sticky ends by the base complementary pairing principle;

(2) preparation of dendritic nucleic acid network nanostructure: equivalently mixing the multi-branched nucleic acid nano structure with the complementary nucleic acid sequence adhesive tail end obtained in the step (1), wherein the mixing temperature is 4-40 ℃, the vibration rotating speed is 0-3000 rpm, and the reaction time is 0.5 h-3 d, so as to prepare a dendritic nucleic acid network nano structure;

(3) preparation of multifunctional nucleic acid-based hybrid nanogel: and (3) mixing the dendritic nucleic acid network nano structure obtained in the step (2) and the micromolecule or the macromolecular compound of the polyphenol hydroxyl according to a proper proportion, standing at room temperature, and preparing the multifunctional nucleic acid-based hybrid nano gel.

The preparation of the multifunctional nucleic acid-based hybrid nanogel can adopt the method. The above-described method is disclosed in order to enable a person skilled in the art to practice the invention, but is not intended to limit the invention in any way.

The invention will be further described with reference to specific embodiments and the accompanying drawings.