CN112851935A

CN112851935A - Active oxygen triggered charge annihilation type super-charge cationic polymer transfection reagent and preparation and application thereof

Info

Publication number: CN112851935A
Application number: CN202110033430.3A
Authority: CN
Inventors: 丁杨; 周建平; 程皓; 张华清; 盛钰
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-01-11
Filing date: 2021-01-11
Publication date: 2021-05-28

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to an active oxygen triggered charge annihilation type super-charge cationic polymer transfection reagent and preparation and application thereof. The transfection reagent consists of a super-charge cationic polymer and a transfection reagent working solution, wherein the super-charge cationic polymer is an aliphatic amine polymer and is obtained by modifying and quaternizing a poly-amino polymer through an active oxygen response group. The super-charge cationic polymer transfection reagent prepared by the invention has high charge density and can provide high-quantity loading of biomacromolecule drugs; under the action of active oxygen, the super-charge cationic polymer is quickly oxidized and hydrolyzed, the positive charges are annihilated, the compressed medicine is quickly released, and the transfection efficiency is high. The transfection reagent is suitable for transfection of nucleic acid, polypeptide and protein biomacromolecules and derivatives thereof, and has the advantages of high loading capacity, small reagent dosage, low toxicity, simple operation, low raw material cost and simple preparation process.

Description

Active oxygen triggered charge annihilation type super-charge cationic polymer transfection reagent and preparation and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an active oxygen triggered charge annihilation type super-charge cationic polymer transfection reagent and preparation and application thereof.

Background

Biological drugs mainly comprise nucleic acid, polypeptide, protein and the like, and have high specificity and target diversity due to the therapeutic action, so that the biological drugs are widely concerned in the treatment of major diseases such as malignant tumors, cardiovascular and cerebrovascular diseases, neurodegenerative diseases and the like in recent years, and are one of the most promising drug research and development fields in the 21 st century. Although biological drugs have many advantages in terms of curative effect, the inherent defects of poor stability, cell uptake barrier and the like severely limit the clinical application of the biological drugs. Therefore, the development of efficient biomolecule packaging and delivery vehicles, and the realization of targeted delivery of biomolecules is the key to promote the development of biopharmaceuticals.

Currently, in biological drug delivery carriers, cationic liposomes have been studied more mature and are gradually applied clinically, for example, the first siRNA drug inpattro sold in the world in 2018 adopts cationic liposomes as a delivery system. At present, in order to

The typical cationic liposome dominates the market of transfection reagents, and the lipid materials, especially modified lipid, have complex acquisition process, so the price is high, the lipid materials are easy to deteriorate, the storage condition requirement is high, and a lot of inconvenience is brought in use. The cationic polymer is a biological drug delivery carrier with great potential, has high positive charge density, can be tightly combined with electronegative biological molecules and compresses the biological molecules to form nano particles, and effectively protects the structural integrity and the biological activity of biological drugs; and the nanoparticles can be taken up by cells by charge interaction with the cell membrane. In addition, the cationic polymer has simple preparation process and stable property, can effectively reduce the cost of the transfection reagent, and simplifies the application method. However, the existing cationic polymer carriers still have certain disadvantages: common cationic polymers such as polyethyleneimine, polylysine, polyenepolyamine, chitosan and the like are rich in amino structures, the charge density of the polymers is greatly influenced by the pH of the environment, and rapid biological drug release in cells is difficult to realize; lack of effective degradation mechanism, excessive positive charge after compressing biological drugs often induces cytotoxicity and the like. Therefore, there is a need in the art to develop a biomolecule transfection reagent that is efficient, low in toxicity, low in cost, and easy to use.

Disclosure of Invention

The invention aims to overcome the defects of the prior transfection technology and provides an active oxygen-triggered charge annihilation type super-charge cationic polymer and application thereof.

The invention also aims to provide an active oxygen-triggered charge annihilation type super-charge cationic polymer transfection reagent and application thereof.

It is still another object of the present invention to provide a method for preparing the transfection reagent.

Specifically, the invention adopts the following technical scheme:

a super-charge cationic polymer is characterized in that the super-charge cationic polymer is an aliphatic amine polymer and comprises an active oxygen response group and a quaternary ammonium salt structure; the active oxygen responsive group can be oxidized and hydrolyzed under the action of active oxygen and is selected from one or more of aromatic boric acid, aromatic boric acid ester, oxalic acid monoaromatic ester and oxalic acid diarylester; the quaternary ammonium salt structure has an ultra-high density positive charge.

The supercharged cationic polymer is preferably selected from those of any structure in which R is₁、R₂、R₃Independently selected from hydrogen, alkyl or aromatic hydrocarbon with carbon number not more than 20 or R₁、R₂The PAMAM is connected into a ring through a chemical bond, the sum of n or x + y is an integer between 50 and 1000, the algebraic number of the PAMAM is 2.0 to 7.0:

the invention relates to application of a super-charge cationic polymer in preparation of a biomolecule loading and delivery system.

Preferably, the biomacromolecule drug is selected from nucleic acid, polypeptide, protein biomacromolecule and derivatives thereof.

A transfection reagent comprises the super-charge cationic polymer and a transfection reagent working solution.

As a preferred choice of the invention, the transfection reagent working solution is selected from one or more of PBS solution, normal saline, 5% glucose solution and cell culture medium.

A preparation method of an active oxygen-triggered charge annihilation type super-charge cationic polymer transfection reagent comprises the following steps:

(1) converting amino in the aliphatic amine polymer into tertiary amine through reductive amination reaction, and separating and purifying to obtain tertiary aminated aliphatic amine polymer;

(2) under the action of a catalyst, reacting a halogenated active oxygen response group with a tertiary aminated aliphatic amine polymer in a reaction medium in a dark place, separating, purifying, and freeze-drying to obtain the super-charge cationic polymer;

(3) and dissolving the super-charge cationic polymer in the working solution of the transfection reagent, and filtering out bacteria to obtain the transfection reagent.

The aliphatic amine polymer in the step (1) is selected from one of polyethyleneimine, polyenepolyamine, polylysine, chitosan and aminoethylethanolamine dendrimer; the reagent used in the reductive amination reaction comprises organic aldehyde and organic carboxylic acid, and the reaction molar ratio of amine groups in the aliphatic amine polymer to aldehyde groups in the organic aldehyde is 1: (1.5-10), wherein the reaction molar ratio of the amino group in the aliphatic amine polymer to the carboxyl group in the organic carboxylic acid is 1: (2.2-10);

in the step (2), the catalyst is one or more of potassium iodide, anhydrous potassium carbonate and anhydrous sodium carbonate; the reaction medium is one or more of N, N-dimethylformamide, dimethyl sulfoxide, methanol, ethanol and acetonitrile.

Preferably, the organic aldehyde is formaldehyde, the organic acid is formic acid, the catalyst is anhydrous potassium carbonate, and the organic solvent is N, N-dimethylformamide.

The reaction temperature of the reductive amination reaction in the step (1) is 60-100 ℃, and the reaction time is 6-24 hours; the separation and purification method comprises the steps of adding acid liquor into reaction liquid, evaporating the reaction liquid to dryness, dissolving obtained solid in water, adding alkali liquor, separating an organic phase, and drying to obtain the tertiary aminated polyamine polymer.

An active oxygen triggered charge annihilation type super-charge cationic polymer transfection reagent is used for transfection of biological macromolecular drugs, wherein the biological macromolecular drugs comprise nucleic acid, polypeptide, protein biological macromolecules and derivatives thereof.

The application of the transfection reagent comprises the following steps:

(1) dissolving the biological macromolecular drug in physiological solution, adding a transfection reagent, and uniformly mixing through mild mechanical action to obtain a super-charge cationic polymer/biological macromolecular drug compound solution;

(2) the compound solution obtained in the step (1) is used for in vitro cell transfection, or is used for one or more of intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, focal local injection, nasal administration, ocular administration and pulmonary administration.

The invention modifies aliphatic amine polymer containing polyamine group by simple chemical process, so that the amine group is converted into quaternary ammonium salt structure and active oxygen response group is introduced to obtain the super charge cationic polymer. The positive charge density of the polymer is greatly increased, the water solubility is further improved, and the compression performance of the biomacromolecule medicine is enhanced; and active oxygen response groups can be removed under the action of active oxygen, positive charges are annihilated, the disintegration of a transfection compound is promoted, biomacromolecule medicines are quickly released, and high-efficiency transfection is realized.

Compared with the prior art, the invention has the advantages that:

(1) the invention provides a brand-new active oxygen triggered charge annihilation type super-charge cationic polymer transfection reagent and preparation and application thereof, the transfection reagent has simple components, and compared with the published patent (201510656807.5), the super-charge cationic polymer has the advantages of simple and convenient synthesis process, good product specificity, high yield, easy purification, cheap and easily available raw materials, easy preparation and mass production.

(2) The invention converts the prior amino cationic polymer into the quaternary ammonium super-charge cationic polymer, obviously improves the charge density without being influenced by the pH of a medium, and improves the water solubility of the polymer; meanwhile, the compression and protection capability of the biological molecules is enhanced, the super-charge cationic polymer can improve the drug loading capacity of biological macromolecules and reduce the dosage of the polymer.

(3) Active oxygen response groups are introduced into the structure of the super-charge cationic polymer provided by the invention, the groups are easy to spontaneously oxidize and hydrolyze under the action of intracellular active oxygen, and the strong electropositive quaternary ammonium salt structure is converted into a charge-neutral tertiary amine structure, so that charge annihilation in cells is realized. The process promotes the rapid release of biomacromolecule drugs in cells, exerts biological functions and improves the transfection efficiency.

(4) And is commercially available

Compared with the prior art, the super-charge cationic polymer transfection reagent prepared by the invention has higher transfection activity and lower cytotoxicity in cells.

Drawings

FIG. 1 is a drawing showing a polymer a-1 of example 1¹H-NMR nuclear magnetic resonance spectrum;

FIG. 2 is a drawing showing the preparation of polymer a-2 in example 1¹H-NMR nuclear magnetic resonance spectrum;

FIG. 3 is a drawing showing the preparation of polymers a to 3 in example 1¹H-NMR nuclear magnetic resonance spectrum;

FIG. 4 shows the preparation of polymer b-1 from example 2¹H-NMR nuclear magnetic resonance spectrum;

FIG. 5 shows the preparation of polymer b-2 from example 2¹H-NMR nuclear magnetic resonance spectrum;

FIG. 6 is a drawing showing the preparation of polymer b-3 in example 2¹H-NMR nuclear magnetic resonance spectrum;

FIG. 7 is a drawing showing the preparation of polymer c-1 in example 3¹H-NMR nuclear magnetic resonance spectrum;

FIG. 8 is a drawing showing the preparation of polymer c-2 in example 3¹H-NMR nuclear magnetic resonance spectrum;

FIG. 9 is a drawing showing the preparation of polymer c-3 in example 3¹H-NMR nuclear magnetic resonance spectrum;

FIG. 10 is a drawing showing the preparation of polymer d-1 in example 4¹H-NMR nuclear magnetic resonance spectrum;

FIG. 11 is a drawing showing the preparation of polymer d-2 in example 4¹H-NMR nuclear magnetic resonance spectrum;

FIG. 12 shows the preparation of polymer d-3 in example 4¹H-NMR nuclear magnetic resonance spectrum;

FIG. 13 is a drawing showing a modification of the polymer e-1 in example 5¹H-NMR nuclear magnetic resonance spectrum;

FIG. 14 shows the polymer e-2 of example 5¹H-NMR nuclear magnetic resonance spectrum;

FIG. 15 is a drawing showing the preparation of polymer e-3 in example 5¹H-NMR nuclear magnetic resonance spectrum;

FIG. 16 is a graph showing the degradation curve of polymer a-1 in example 6 under the action of active oxygen;

FIG. 17 is the electrophoresis chart of the gel retardation experiment of the polymer a-1/siRNA nanocomposite in example 7;

FIG. 18 is the gel blocking experimental electrophoretogram of PEI/siRNA nanocomplex in example 7;

FIG. 19 shows the particle size and polydispersity index of polymer a-1/siRNA nanocomposites at different N/P in example 8;

FIG. 20 is the surface zeta potential of the polymer a-1/siRNA nanocomplex in example 9;

FIG. 21 is a transmission electron microscope observation of polymer a-1/siRNA nanocomposite in example 10;

FIG. 22 shows the variation of the particle size and the effect of active oxygen for the polymer a-1/siRNA nanocomposite in example 11;

FIG. 23 is the electrophoresis chart of the gel retardation experiment after the action of the polymer a-1/siRNA nanocomposite and active oxygen in example 12;

FIG. 24 is a graph showing the cytotoxicity of Polymer a-1 and Polymer a-1/siRNA complex in example 13;

FIG. 25 is a confocal microscope observation of the cellular uptake of polymer a-1/siRNA nanocomplexes of example 14;

FIG. 26 is a confocal microscope observation of siRNA release in cells by polymer a-1/siRNA nanocomplexes in example 15;

FIG. 27 is a fluorescence observation graph of transfection efficiency of Polymer a-1/siRNA nanocomplexes in example 16;

FIG. 28 is a fluorescence observation graph of transfection efficiency of Polymer b-1/siRNA nanocomplexes in example 17;

FIG. 29 is a fluorescence observation graph of the transfection efficiency of polymer c-1/siRNA nanocomplexes in example 18;

FIG. 30 is a fluorescence observation graph of the transfection efficiency of polymer d-1/siRNA nanocomplexes in example 19;

FIG. 31 is a fluorescence observation graph of transfection efficiency of polymer e-1/siRNA nanocomplexes in example 20.

Detailed Description

The present invention provides some specific examples, but the present invention is not limited by these examples, and the following examples are only used to help understanding the method of the present invention and its core idea. It should be noted that several modifications or improvements of the present invention may be made without departing from the principle of the present invention, and the protection scope of the present invention is also covered by the claims. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention.

The invention discloses an active oxygen triggered charge annihilation type super-charge cationic polymer transfection reagent and preparation and application thereof. The polymer transfection reagent has ultrahigh positive charge density, and can efficiently compress biomacromolecule medicines to form uniform and stable spherical nano particles; the nanometer particle is effectively absorbed by cells through physical adsorption, charge action or cell surface receptor recognition, and annihilates positive charges under the action of Reactive Oxygen Species (ROS) to release biological macromolecule drugs into cytoplasm to play a biological function. The preparation process of the super-charge cationic polymer transfection reagent is simple and easy to implement, the synthetic raw materials are cheap and easy to obtain, the use is simple and convenient, the dosage is small, the transfection activity is high, the cytotoxicity is low, and the application prospect is wide in the fields of biology, medical research and clinical treatment.

Example 1: synthesis of Polymer a

5.75g of formic acid was weighed into a 150mL round bottom flask, and 1.08g of PEI (Mw 10kDa) was slowly added with cooling in an ice-water bath and dissolved well to give a clear solution. Adding 5.62mL of 37% formaldehyde solution, and carrying out reflux reaction for 12h under a heating condition, wherein the reaction temperature is controlled to be 90-100 ℃. And then separating and purifying the reaction product, adding 12.5mL of hydrochloric acid (4mol/L) into the reaction solution, fully mixing uniformly, evaporating to be dry, dissolving the obtained solid in 10mL of water, adding 6.25mL of sodium hydroxide solution (18mol/L), separating an upper organic phase, extracting a lower aqueous phase for 3 times by using 6mL of benzene, combining the organic phases, adding anhydrous sodium carbonate particles to remove water, and performing rotary evaporation to remove benzene to obtain the PEI derivative (ter-PEI) of which all amino groups are tertiary amine. The tertiary aminated PEI derivative was a yellow viscous liquid with a yield of about 90%.

And taking tertiary aminated PEI as an intermediate product, and coupling different active oxygen responsive groups to obtain the polymer a1-a 3.

R is

Is marked asA polymer a-1;

r is

Denoted as polymer a-2;

r is

Denoted as polymer a-3.

Synthesis of Polymer a-1: 30mg of tert-PEI is dissolved in 1.5mL of DMF/methanol (2:1, v/v), 65mg of 4-bromomethylbenzeneboronic acid is added, and the mixture is stirred until the mixture is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (the polymer a-1, n is 80). Polymer a-1 was a white to pale yellow solid powder with a yield of 95%. Of polymers a-1¹The H-NMR spectrum is shown in FIG. 1.

Synthesis of Polymer a-2: 30mg of tert-PEI is dissolved in 1.5mL of DMF/methanol (2:1, v/v), 65mg of 4 mg of pinacol bromomethylbenzoate is added, and the mixture is stirred until the pinacol bromomethylbenzoate is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer a-2, n is 80). Polymer a-2 was a white solid powder with a yield of 91%. Of polymers a-2¹The H-NMR spectrum is shown in FIG. 2.

Synthesis of Polymer a-3: (1) synthesizing an active oxygen response group intermediate methyl oxalate (4-bromomethyl) phenyl ester. Dissolving 65mg of 4-bromomethylbenzeneboronic acid in 3mL of DMF, adding a hydrogen peroxide solution to a final concentration of 10mM, and reacting for 24 hours in a dark place; heating the reaction solution to 100-120 ℃, and reacting for 6h in a dark place; removing water in the reaction liquid through rotary evaporation, adding 70mg of dimethyl oxalate, adding a proper amount of sulfuric acid for catalysis, and reacting for 24 hours in a dark place; to obtain the oxalic acid methyl ester (4-bromomethyl) phenyl ester. (2) 30mg of tert-PEI is dissolved in 1.5mL of DMF/methanol (2:1, v/v), the product of (1) is added to the reaction solution, and a proper amount of anhydrous Na is added₂CO₃Adding small amount of KI, and keeping out lightThe reaction was carried out at room temperature for 24 hours. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer a-3, n is 80). Polymer a-3 was a white crystalline solid in 87% yield. Of polymers a-3¹The H-NMR spectrum is shown in FIG. 3.

Example 2: synthesis of Polymer b

4.60g of formic acid was weighed into a 150mL round-bottomed flask, and 2.56g of polylysine was slowly added with cooling in an ice water bath and fully dissolved to give a clear solution. Adding 8.19mL of 37% formaldehyde solution, and carrying out reflux reaction for 12h under a heating condition, wherein the reaction temperature is controlled to be 80-90 ℃. And then, separating and purifying a reaction product, adding 12.5mL of hydrochloric acid (4mol/L) into a reaction solution, fully and uniformly mixing, evaporating to be dry, dissolving the obtained solid in 10mL of water, adding 6.25mL of sodium hydroxide solution (18mol/L), separating an upper organic phase, extracting a lower aqueous phase for 3 times by using 6mL of benzene, combining the organic phases, adding anhydrous sodium carbonate particles to remove water, and performing rotary evaporation to remove benzene to obtain the polylysine derivative with all amino groups being tertiary amine. The tertiary aminated polylysine was a yellow powder solid in about 80% yield.

R is

Denoted as polymer b-1;

r is

Denoted as polymer b-2;

r is

Denoted as polymer b-3.

Synthesis of Polymer b-1: 156.19mg of tertiary aminated polylysine is dissolved in 6mL of DMF/methanol (2:1, v/v), 215mg of 4-bromomethylbenzeneboronic acid is added, and the mixture is stirred until the mixture is completely dissolved; adding intoA small amount of KI is added, and the mixture is stirred and reacted for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer b-1, n is 100). Polymer b-1 was a white to pale yellow solid powder in 96% yield. Of polymer b-1¹The H-NMR spectrum is shown in FIG. 4.

Synthesis of Polymer b-2: 156.19mg of tertiary amination polylysine is dissolved in 6mL of DMF/methanol (2:1, v/v), 297mg of 4-bromomethylbenzeneboronic acid pinacol ester is added, and the mixture is stirred until the mixture is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely, the active oxygen-triggered charge annihilation-type super-charge cationic polymer (polymer b2, n is 100). Polymer b-2 was a white to pale yellow solid powder in 92% yield. Of polymers b-2¹The H-NMR spectrum is shown in FIG. 5.

Synthesis of Polymer b-3: (1) synthesizing an active oxygen response group intermediate methyl oxalate (4-bromomethyl) phenyl ester. Dissolving 215mg of 4-bromomethylbenzeneboronic acid in 10mL of DMF, adding a hydrogen peroxide solution to a final concentration of 10mM, and reacting for 24 hours in a dark place; heating the reaction solution to 100-120 ℃, and reacting for 6h in a dark place; removing water in the reaction liquid through rotary evaporation, adding 118mg of dimethyl oxalate, adding a proper amount of sulfuric acid for catalysis, and reacting for 24 hours in a dark place; to obtain the oxalic acid methyl ester (4-bromomethyl) phenyl ester. (2) 156.19mg of tert-PEI was dissolved in 6mL of DMF/methanol (2:1, v/v), the product of (1) was added to the reaction mixture, and an appropriate amount of anhydrous Na was added₂CO₃And adding a small amount of KI, and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer b-3, n is 100). Polymer b-3 was a white solid powder with a yield of 90%. Of polymers b-3¹The H-NMR spectrum is shown in FIG. 6.

Example 3: synthesis of Polymer c

4.60g of formic acid was weighed into a 150mL round-bottom flask, and 0.86g of polyene polyamine was slowly added under cooling in an ice-water bath and sufficiently dissolved to obtain a clear solution. Adding 8.19mL of 37% formaldehyde solution, and carrying out reflux reaction for 12h under a heating condition, wherein the reaction temperature is controlled to be 80-90 ℃. And then separating and purifying a reaction product, adding 12.5mL of hydrochloric acid (4mol/L) into a reaction solution, fully and uniformly mixing, evaporating to be dry, dissolving the obtained solid in 10mL of water, adding 6.25mL of sodium hydroxide solution (18mol/L), separating an upper organic phase, extracting a lower aqueous phase for 3 times by using 6mL of benzene, combining the organic phases, adding anhydrous sodium carbonate particles to remove water, and performing rotary evaporation to remove benzene to obtain the polyene polyamine derivative of which all amino groups are tertiary amines. The tertiary aminated polyene polyamine is a yellow viscous liquid with a yield of about 90%.

R is

Denoted as polymer c-1;

r is

Denoted as polymer c-2;

r is

Denoted as polymer c-3.

Synthesis of Polymer c-1: dissolving 57.06mg of tertiary aminated polyene polyamine in 4.5mL of DMF/methanol (2:1, v/v), adding 215mg of 4-bromomethylbenzeneboronic acid, and stirring until the mixture is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely, the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer c-1, x is 100, and y is 100). Polymer c-1 was a white to pale yellow solid powder in 95% yield. Of polymer c-1¹The H-NMR spectrum is shown in FIG. 7.

Synthesis of Polymer c-2: dissolving 57.06mg of tertiary aminated polyene polyamine in 4.5mL of DMF/methanol (2:1, v/v), adding 297mg of pinacol 4-bromomethylbenzoate, and stirring until the materials are completely dissolved; addingAdding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely, the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer c-2, x is 100, and y is 100). Polymer c-2 was a white to pale yellow solid powder in 95% yield. Of polymer c-2¹The H-NMR spectrum is shown in FIG. 8.

Synthesis of Polymer c-3: (1) synthesizing an active oxygen response group intermediate methyl oxalate (4-bromomethyl) phenyl ester. Dissolving 215mg of 4-bromomethylbenzeneboronic acid in 10mL of DMF, adding a hydrogen peroxide solution to a final concentration of 10mM, and reacting for 24 hours in a dark place; heating the reaction solution to 100-120 ℃, and reacting for 6h in a dark place; removing water in the reaction liquid through rotary evaporation, adding 118mg of dimethyl oxalate, adding a proper amount of sulfuric acid for catalysis, and reacting for 24 hours in a dark place; to obtain the oxalic acid methyl ester (4-bromomethyl) phenyl ester. (2) Dissolving 57.06mg of tertiary aminated polyene polyamine in 4.5mL of DMF/methanol (2:1, v/v), adding the product obtained in step (1) into the reaction solution, and adding a proper amount of anhydrous Na₂CO₃And adding a small amount of KI, and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction solution is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely, the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer c-3, x is 100, and y is 100). Polymer c-3 was a white to pale yellow solid powder in 95% yield. Of polymer c-3¹The H-NMR spectrum is shown in FIG. 9.

Example 4: synthesis of Polymer d

Synthesis of Polymer d: 4.60g of formic acid was weighed into a 150mL round-bottom flask, and 3.22g of chitosan was slowly added with cooling in an ice-water bath and fully dissolved to obtain a clear solution. Adding 8.19mL of 37% formaldehyde solution, and carrying out reflux reaction for 12h under a heating condition, wherein the reaction temperature is controlled to be 90-100 ℃. And then, separating and purifying a reaction product, adding 12.5mL of hydrochloric acid (4mol/L) into a reaction solution, fully and uniformly mixing, evaporating to be dry, dissolving the obtained solid in 10mL of water, adding 6.25mL of sodium hydroxide solution (18mol/L), separating an upper organic phase, extracting a lower aqueous phase for 3 times by using 6mL of benzene, combining the organic phases, adding anhydrous sodium carbonate particles to remove water, and performing rotary evaporation to remove benzene to obtain the chitosan derivative with all amino groups being tertiary amine. The tertiary aminated chitosan was a yellow solid in about 93% yield.

R is

Denoted as polymer d-1;

r is

Denoted as polymer d-2;

r is

Denoted as polymer d-3.

Synthesis of Polymer d-1: 189.23mg of tertiary amination chitosan is dissolved in 12mL of DMF/methanol (2:1, v/v), 215mg of 4-bromomethylbenzeneboronic acid is added, and the mixture is stirred until the mixture is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction liquid is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (the polymer d-1, n is 790). Polymer d-1 was a white to pale yellow solid powder in 98% yield. Of polymer d-1¹The H-NMR spectrum is shown in FIG. 10.

Synthesis of Polymer d-2: 189.23mg of tertiary amination chitosan is dissolved in 12mL of DMF/methanol (2:1, v/v), 297mg of 4-bromomethylbenzeneboronic acid pinacol ester is added, and the mixture is stirred until the tertiary amination chitosan is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction liquid is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (the polymer d-2, n is 790). Polymer d-2 was a white to pale yellow solid powder in 92% yield. Of polymer d-2¹The H-NMR spectrum is shown in FIG. 11.

Synthesis of Polymer d-3: (1) synthesizing an active oxygen response group intermediate methyl oxalate (4-bromomethyl) phenyl ester. 215mg of 4-bromomethylbenzene are takenDissolving boric acid in 10mL of DMF, adding a hydrogen peroxide solution to a final concentration of 10mM, and reacting for 24 hours in a dark place; heating the reaction solution to 100-120 ℃, and reacting for 6h in a dark place; removing water in the reaction liquid through rotary evaporation, adding 118mg of dimethyl oxalate, adding a proper amount of sulfuric acid for catalysis, and reacting for 24 hours in a dark place; to obtain the oxalic acid methyl ester (4-bromomethyl) phenyl ester. (2) 189.23mg of tertiary aminated chitosan is dissolved in 12mL of DMF/methanol (2:1, v/v), the product obtained in the step (1) is added into the reaction solution, and a proper amount of anhydrous Na is added₂CO₃And adding a small amount of KI, and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, the reaction liquid is collected, dialyzed in ultrapure water for 48h, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type super-charge cationic polymer (polymer d-3, n is 790). Polymer d-3 was a white to pale yellow solid powder in 86% yield. Of polymer d-3¹The H-NMR spectrum is shown in FIG. 12.

Example 5: synthesis of Polymer e

Synthesis of Polymer e: weighing 4.60g formic acid in a 150mL round bottom flask, slowly adding 2.3g PAMAM dendrimer (3 rd generation) while cooling in ice water bath, and fully dissolving to obtain a clear solution. Adding 8.19mL of 37% formaldehyde solution, and carrying out reflux reaction for 12h under a heating condition, wherein the reaction temperature is controlled to be 90-100 ℃. And then, separating and purifying a reaction product, adding 12.5mL of hydrochloric acid (4mol/L) into a reaction solution, fully and uniformly mixing, evaporating to be dry, dissolving the obtained solid in 10mL of water, adding 6.25mL of sodium hydroxide solution (18mol/L), separating an upper organic phase, extracting a lower aqueous phase for 3 times by using 6mL of benzene, combining the organic phases, adding anhydrous sodium carbonate particles to remove water, and performing rotary evaporation to remove benzene to obtain the PAMAM derivative with all amino groups being tertiary amine. The tertiary aminated PAMAM was an orange viscous liquid with a yield of about 91%.

R is

Denoted as polymer e-1;

r is

Denoted as polymer e-2;

r is

Denoted as polymer e-3.

Synthesis of Polymer e-1: 143.19mg of tertiary aminated PAMAM is dissolved in 8mL of DMF/methanol (2:1, v/v), 215mg of 4-bromomethylbenzeneboronic acid is added, and the mixture is stirred until the mixture is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, reaction liquid is collected, dialyzed in ultrapure water for 48 hours, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type supercharged cationic polymer (the polymer e-1, PAMAM dendrimer is the 3 rd generation). Polymer e-1 was a white to pale yellow solid powder in 88% yield. Of Polymer e-1¹The H-NMR spectrum is shown in FIG. 13.

Synthesis of Polymer e-2: 143.19mg of tertiary amination PAMAM is dissolved in 8mL of DMF/methanol (2:1, v/v), 297mg of 4-bromomethylbenzeneboronic acid pinacol ester is added, and the mixture is stirred until the mixture is completely dissolved; adding a small amount of KI, stirring and reacting for 24 hours at room temperature in a dark place. After the reaction is finished, reaction liquid is collected, dialyzed in ultrapure water for 48 hours, and freeze-dried to collect the final product, namely the active oxygen-triggered charge annihilation type supercharged cationic polymer (the polymer e-2, the PAMAM dendrimer is the 3 rd generation). Polymer e-2 was a white to pale yellow solid powder in 96% yield. Of polymer e-2¹The H-NMR spectrum is shown in FIG. 14.

Synthesis of Polymer e-3: (1) synthesizing an active oxygen response group intermediate methyl oxalate (4-bromomethyl) phenyl ester. Dissolving 215mg of 4-bromomethylbenzeneboronic acid in 10mL of DMF, adding a hydrogen peroxide solution to a final concentration of 10mM, and reacting for 24 hours in a dark place; heating the reaction solution to 100-120 ℃, and reacting for 6h in a dark place; removing water in the reaction liquid through rotary evaporation, adding 118mg of dimethyl oxalate, adding a proper amount of sulfuric acid for catalysis, and reacting for 24 hours in a dark place; to obtain the oxalic acid methyl ester (4-bromomethyl) phenyl ester. (2) 143.19mg of tertiary aminated PAMAM was dissolved in 8mL of DMF/methanol (2:1, v/v), the product of (1) was added to the reaction mixture, and a suitable amount of anhydrous Na was added₂CO₃And addingA small amount of KI was reacted at room temperature for 24 hours under exclusion of light. After the reaction is finished, reaction liquid is collected, dialyzed in ultrapure water for 48 hours, and freeze-dried to collect the final product, namely, the active oxygen-triggered charge annihilation type supercharged cationic polymer (polymer e-3, PAMAM dendrimer is the 3 rd generation). Polymer e-3 was a white to pale yellow solid powder in 87% yield. Of polymer e-3¹The H-NMR spectrum is shown in FIG. 15.

Example 6: ROS-triggered degradation of Polymer a-1

Under the action of ROS, oxidizing and dropping a phenylboronic acid structural unit of the super-charge polymer a-1 to generate 4-Hydroxymethyphenol (HMP), and observing the degradation kinetics of the polymer a-1 by monitoring the degradation product in real time, wherein the method comprises the following specific steps:

preparing a standard solution: accurately weighing 2mg of p-hydroxybenzyl alcohol standard substance, and diluting to 10mL by 10% methanol water solution to obtain 200 μ g/mL stock solution. And (4) diluting the standard substance stock solution according to a certain multiple gradient to obtain a series of concentration standard samples.

Sample preparation: polymer a-1 was dissolved in 1mM H₂O₂Obtaining 0.3mg/mL solution, and detecting 20 mu L sample by high performance liquid chromatography in 10, 20, 40, 60, 90, 120, 180 and 300 min. The liquid phase conditions are as follows: waters symmetry C18 reversed phase column (4.6 mm. times.150 mm, 5 μm), mobile phase:

10% methanol-water solution, detection wavelength: 220nm, flow rate: 1mL/min, column temperature: 37 ℃ is carried out. And (3) analyzing the peak area of the p-hydroxybenzyl alcohol in 3.5min by taking a standard curve of the concentration of the standard substance to the absorption peak area, and calculating the degradation rate of the polymer a-1. The detection result is shown in FIG. 16, and the experiment proves that the degradation rate of the polymer a-1 can reach 80% within 1h under the action of ROS.

Example 7: biomolecule compressibility of Polymer a-1

Weighing a proper amount of polymer a-1, dissolving the polymer a-1 in ultrapure water to prepare 10mg/mL polymer a-1 solution, and filtering for later use. siRNA is dissolved by RNAse-free water to prepare a siRNA solution with the concentration of 100 mu g/mL. Diluting the polymer a-1 solution into series of concentrations corresponding to different nitrogen-phosphorus ratios, mixing the polymer a-1 solution with the siRNA solution in a vortex mode with the volume of 1:2, and standing at room temperature for more than 30min to obtain the polymer a-1/siRNA nanoparticles corresponding to the series of nitrogen-phosphorus ratios (N/P). Similarly, PEI/siRNA complexes were prepared with PEI by vortex mixing according to the corresponding N/P values.

Accurately weighing 2g of agarose in a conical flask, adding 100mL of 1 XTAE nucleic acid electrophoresis buffer solution, stirring and dispersing uniformly, heating by microwave to obtain a clear and transparent agarose solution, adding ultrapure water to scale, and preparing a 2% agarose solution; after the solution was cooled to about 60 ℃, 3 μ L of Goldview was added and mixed well, and the agarose solution was poured into the gel plate. After the gel is completely cooled, the agarose gel is placed in a horizontal electrophoresis tank, and 500mL of 1 XTAE nucleic acid electrophoresis buffer is added to fill the electrophoresis tank and to submerge the gel by about 2 mm. A sample of 20. mu.L of the polymer a-1/siRNA nanoparticle sample was mixed with 5. mu.L of 5 × Loading buffer, 20. mu.L of the mixture was loaded into a well, and electrophoresis was carried out at 90V for 30 min. And after the electrophoresis is finished, placing the gel in an ultraviolet gel imager for imaging and shooting. The experimental results of the polymer a-1 and the PEI are respectively shown in FIGS. 17 and 18, and the polymer a-1 can realize effective siRNA compression and retard the migration of siRNA in gel under the condition that the N/P of the polymer a-1 is more than or equal to 1.5; and PEI can realize the complete compression of siRNA under the condition that N/P is more than or equal to 2. The main reason for this is that the PEI structure is positively charged by protonation of the N therein, wherein the main protonatable structures are primary and secondary amines, while the tertiary amines, which account for the total number of amines 1/3, are not easily protonated, so that the positive charge density of polymer a-1 is about 1/2 higher than that of PEI. The cationic polymer provided in CN105153339A can realize complete compression of nucleic acid under the condition of N/P ≧ 3, while the nucleic acid compression efficiency of the super-charge cationic polymer provided by the invention is about 2 times, and the polymer dosage in application can be reduced by about 50% due to the higher charge density of the polymer a-1 of the invention compared with compound 1 in CN 105153339A.

Example 8: particle size distribution of Polymer a-1/siRNA complexes

Polymer a-1/siRNA nanocomposites were prepared according to a series of N/P values (2, 3, 4, 5, 6, 7, 8) and their nanoparticle size distribution was determined by dynamic light scattering technique at 25 ℃ with 3 tests per group of samples. As shown in FIG. 19, the size of the polymer a-1/siRNA nanocomplexes was about 50nm and the distribution was uniform at an N/P value of 4.

Example 9: surface electrical properties of Polymer a-1/siRNA complexes

And (3) taking a freshly prepared polymer a-1/siRNA nano-composite with the N/P value of 4, and measuring the surface zeta potential of the polymer a1/siRNA nano-composite by using a dynamic light scattering instrument. As shown in FIG. 20, the zeta potential of the surface of the polymer a-1/siRNA nano-complex is about +20 mV.

Example 10: morphological characterization of Polymer a-1/siRNA nanocomposites

And (3) taking a piece of copper mesh by using a pair of tweezers, immersing the copper mesh into the polymer a-1/siRNA nano-composite solution, taking out the copper mesh, dropwise adding a 2.0% phosphotungstic acid solution on the copper mesh, dyeing for 15s, sucking away the phosphotungstic acid by using filter paper, and placing the sample under a transmission electron microscope to observe the morphological characteristics of the sample after the copper mesh is completely dried. As shown in FIG. 21, the polymer a-1/siRNA nano-composite has a sphere-like structure, a particle size of about 45nm, a uniform distribution, and a measurement result according to a dynamic light scattering method.

Example 11: ROS responsiveness of Polymer a-1/siRNA nanocomposites

Dispersing freshly prepared polymer a-1/siRNA nano-composite into a series of concentrations H₂O₂The solution (0, 0.5, 1, 2, 5, 10mM) was incubated at 37 ℃ for 6 hours, and the change in particle size of the polymer a-1/siRNA complex was measured by dynamic light scattering. As shown in FIG. 22, it can be seen that the particle size of the polymer a-1/siRNA nanocomposite is significantly increased under the action of ROS, indicating that under the action of reactive oxygen, the positive charge of the polymer a-1 is rapidly reduced, the gene drug cannot be tightly compressed, and the nanocomposite gradually disintegrates.

Example 12: ROS-triggered release of siRNA in Polymer a-1/siRNA nanocomposites

Accurately weighing 2g of agarose in a conical flask, adding 100mL of 1 XTAE nucleic acid electrophoresis buffer solution, stirring and dispersing uniformly, heating by microwave to obtain a clear and transparent agarose solution, adding ultrapure water to scale, and preparing a 2% agarose solution; after the solution was cooled to about 60 ℃, 3 μ L of Goldview was added and mixed well, and the agarose solution was poured into the gel plate. After the gel is completely cooled, the agarose gel is placed in a horizontal electrophoresis tank, and 500mL of 1 XTAE nucleic acid electrophoresis buffer is added to fill the electrophoresis tank and to submerge the gel by about 2 mm.

Dispersing freshly prepared polymer a-1/siRNA nano-composite into a series of concentrations H₂O₂The cells were incubated in solution (0, 0.5, 1, 1.5, 2.5, 5, 10mM) at 37 ℃ for 12 h. Uniformly mixing each sample with a Loading buffer, Loading the sample, and carrying out electrophoresis for 20min under the conditions that the voltage is 90V and the current is 80 mA; with free siRNA and H₂O₂The solution co-incubated samples served as positive controls. And after the electrophoresis is finished, placing the gel in an ultraviolet gel imager for imaging and photographing. The detection result is shown in figure 23, the free siRNA is released after the polymer a-1/siRNA nano compound reacts with 1mM ROS for 12h, and the gene drug is released obviously under the condition that the concentration of ROS is more than or equal to 1.5 mM.

Example 13: evaluation of safety of Polymer a-1

The cytotoxicity of the polymer a-1 and the polymer a-1/siRNA complex was examined by MTT method to evaluate the safety of the polymer a-1. After the cells are cultured to logarithmic phase, the cells are digested by pancreatin, collected by centrifugation and inoculated in a 96-well plate with a cell density of about 5X 10³One/well, 5% CO at 37 ℃₂Culturing in incubator for 12 h. The original medium was discarded and serum-free medium containing different concentrations of Polymer a-1, Polymer a-1/siRNA (concentration of Polymer a was 0.002, 0.02, 0.2, 2, 20, 200. mu.g/mL) was added. After an additional 48h of incubation, the medium was replaced with fresh medium and 20. mu.L of MTT (5mg/mL) solution was added to each well and incubation continued for 4h at 37 ℃ in an incubator containing 5% CO 2. The medium was discarded and 100 μ L DMSO added to dissolve formazan crystals produced in viable cells. Meanwhile, setting blank and negative control holes, wherein the blank control hole is a solvent without cells, the negative control is only cells and DMSO, each group is repeated for 5 times, measuring the absorbance value at 570nm by using an enzyme-labeling instrument, and calculating the cell activity according to the formula (1).

As shown in FIG. 24, when the concentration of the polymer a-1 is in the range of 0-20. mu.g/mL, the cell viability can be maintained at more than 90%, indicating that the polymer a-1 has good biological safety.

Example 14: cellular uptake of Polymer a-1/siRNA nanocomposites

The study on the cellular uptake behavior of the polymer a-1/siRNA nanocomposite was performed by a laser confocal microscope. Polymer a-1/Cy5-siRNA nanocomplexes were prepared using Cy 5-labeled siRNA (Cy 5-siRNA). Digesting with pancreatin when the cells grow to logarithmic phase, centrifuging at 800rpm for 5min, collecting cells, and mixing the cells at 1 × 10⁴The density of each well is inoculated in a 24-well plate and a laser confocal culture dish, the temperature is 37 ℃, and the CO content is 5 percent₂Experiments were performed after overnight incubation in an incubator. The original culture medium is aspirated, PBS is added to wash the cells carefully for 2 times, and the polymer a-1/Cy5-siRNA nano-complex is diluted and added to the corresponding well by adopting a DMEM culture medium without serum. The final concentration of Cy5-siNC per well was 20nM in a volume of 0.5 mL. The cells were placed at 37 ℃ with 5% CO₂Incubating in an incubator for 1, 2, 4 and 8 hours. For the laser confocal microscope samples, the cells were fixed with 4% paraformaldehyde solution for 20min at different time points, the cell nuclei were stained with DAPI, and the uptake of the polymer a-1/Cy5-siRNA nanocomposite by the cells was observed under the laser confocal microscope. As shown in FIG. 25, the polymer a-1/Cy5-siRNA nano-complex can be efficiently taken up by cells, and abundant phenylboronic acid groups on the surface of the complex may participate in the process of promoting cellular uptake.

Example 15: release of intracellular siRNA from Polymer a-1/siRNA nanocomposites

And observing the positioning of the carrier and the gene drug in the cell by a laser confocal microscope, and inspecting the gene release behavior in the cell.

Synthesis of fluorescent labeling carrier: weighing PEI 129mg dissolved in 2mL H₂In O, 37.6mg of 5(6) -carboxyfluorescein succinimidyl ester is dissolved in 200 mu L H₂And O, dropwise adding the mixture into a PEI aqueous solution, dialyzing to remove free 5(6) -carboxyl fluorescein succinimide ester after reacting for 12 hours, and freeze-drying to collect a product. Synthesis of F Using the Synthesis route for Polymer a-1AM fluorescent-labeled Polymer a-1 (FAM-Polymer a-1).

And preparing a double-fluorescence-labeled nano-complex by using the FAM-labeled polymer a-1 and Cy 5-siRNA. Laying logarithmic phase growth cells in a laser confocal culture dish, removing the original culture medium after the cells adhere to the wall, replacing the original culture medium with a serum-free culture medium containing FAM-polymer a-1/Cy5-siRNA nano-composite at 37 ℃ and 5% CO₂After culturing for 4h in the incubator, discarding the serum-free culture medium containing the nano-composite, and replacing a fresh serum-free culture medium to continue culturing for 6 h. Cells were fixed with 4% paraformaldehyde at 0.5, 1, 3, 6h, respectively, and nuclei were stained with DAPI. And (3) observing the intracellular distribution of the gene drug and the carrier at different time points by adopting a laser confocal microscope to explore the release behavior of the intracellular gene drug. The experimental result is shown in fig. 26, as the time is prolonged, the gene vector indicated by green and the gene drug indicated by red fluorescence are gradually separated and are basically and completely separated after 6h, which shows that the polymer a-1/siRNA nano-composite can realize the rapid release of the gene drug under the action of ROS after entering the cell, is beneficial to the gene drug to exert the biological function and improves the transfection efficiency of the gene drug.

Example 16: cell transfection Activity of Polymer a-1/siRNA nanocomposites

Preparing a polymer a-1/siEGFP nano-composite by using the polymer a-1 and siRNA (siEGFP) targeting EGFP protein; polymer a-1/siNC nanocomposites were prepared with Polymer a-1 and negative control siRNA (siNC) as controls.

Taking 4T1-GFP cells stably expressing GFP in logarithmic growth phase, collecting cells by trypsinization, and performing 1 × 10 cell selection⁴The cells were plated at a density of one well in 24-well plates and the total volume of the medium was 0.5 mL. 5% CO at 37 ℃₂Incubate overnight. The original medium was discarded and the cells were washed, replaced with serum-free medium containing polymer a-1/siEGFP, polymer a-1/siNC nanocomposite, untreated 4T1-GFP cells were used as a blank control, and the cells were treated with serum-free medium containing 10mM antioxidant N-acetyl-L-cysteine (NAC) and polymer a-1/siEGFP, all at siRNA concentrations of 100 nM. Cells were incubated at 37 ℃ with 5% CO₂The culture was continued for 8h in an incubator with replacement of fresh serum-free medium, wherein NAC-treated cells were cultured for 24h with serum-free medium containing 10mM NAC. The expression level of the reporter gene GFP was observed under an inverted fluorescence microscope and a photograph was taken. The experimental results are shown in FIG. 27, the polymer a-1/siNC nano-complex does not affect the GFP expression level in the cells, and the treatment of the polymer a-1/siEGFP produces a remarkable GFP expression inhibition rate; in cells after NAC co-treatment, the expression inhibition rate of GFP is obviously reduced. Experimental results show that the polymer a-1 can be used for high-efficiency transfection of siRNA, and ROS-triggered gene release plays a key role in improving gene transfection efficiency and cell level selective gene silencing.

Example 17: cell transfection Activity of Polymer b-1/siRNA nanocomposites

Preparing a polymer b-1/siEGFP nano-composite by using the polymer b-1 and siRNA (siEGFP) targeting EGFP protein; polymer b-1/siNC nanocomposites were prepared with Polymer b-1 and negative control siRNA (siNC) as controls.

Taking 4T1-GFP cells stably expressing GFP in logarithmic growth phase, collecting cells by trypsinization, and performing 1 × 10 cell selection⁴The cells were plated at a density of one well in 24-well plates and the total volume of the medium was 0.5 mL. 5% CO at 37 ℃₂Incubate overnight. The original medium was discarded and the cells were washed, replaced with serum-free medium containing polymer b-1/siEGFP, polymer b-1/siNC nanocomposite, untreated 4T1-GFP cells were used as a blank control, and in addition, the cells were treated with serum-free medium containing 10mM antioxidant N-acetyl-L-cysteine (NAC) and polymer b-1/siEGFP, both siRNA concentrations being 100 nM. Cells were incubated at 37 ℃ with 5% CO₂The culture was continued for 8h in an incubator with replacement of fresh serum-free medium, wherein NAC-treated cells were cultured for 24h with serum-free medium containing 10mM NAC. The expression level of the reporter gene GFP was observed under an inverted fluorescence microscope and a photograph was taken. The experimental result is shown in FIG. 28, compared with untreated 4T1-GFP cells, the polymer b-1/siNC nano-composite does not affect the GFP expression level in the cells, the polymer b-1/siEGFP obviously inhibits the GFP expression, and the transfection efficiency of the polymer b-1/siEGFP is obvious after intracellular ROS is eliminated by NACAnd decreases. Experimental results show that the polymer b-1 can be used for high-efficiency transfection of siRNA, and ROS-triggered gene release plays a key role in improving gene transfection efficiency and cell level selective gene silencing.

Example 18: cell transfection Activity of Polymer c-1/siRNA nanocomposites

Preparing a polymer c-1/siEGFP nano-composite by using the polymer c-1 and siRNA (siEGFP) targeting EGFP protein; polymer c-1/siNC nanocomposites were prepared with Polymer c-1 and negative control siRNA (siNC) as controls.

Taking 4T1-GFP cells stably expressing GFP in logarithmic growth phase, collecting cells by trypsinization, and performing 1 × 10 cell selection⁴The cells were plated at a density of one well in 24-well plates and the total volume of the medium was 0.5 mL. 5% CO at 37 ℃₂Incubate overnight. The original medium was discarded and the cells were washed and replaced with serum-free medium containing polymer c-1/siEGFP, polymer c-1/siNC nanocomposite, untreated 4T1-GFP cells were used as a blank control, and in addition, cells were treated with serum-free medium containing 10mM antioxidant N-acetyl-L-cysteine (NAC) and polymer c-1/siEGFP, with siRNA concentrations of each group being 100 nM. Cells were incubated at 37 ℃ with 5% CO₂The culture was continued for 8h in an incubator with replacement of fresh serum-free medium, wherein NAC-treated cells were cultured for 24h with serum-free medium containing 10mM NAC. The expression level of the reporter gene GFP was observed under an inverted fluorescence microscope and a photograph was taken. The experimental results are shown in FIG. 29, compared with untreated 4T1-GFP cells, the GFP expression level of 4T1-GFP cells is not affected by the polymer c-1/siNC nano compound, GFP expression is remarkably inhibited by the polymer c-1/siEGFP, and the transfection efficiency of the polymer c-1/siEGFP is remarkably reduced after intracellular ROS are eliminated by NAC. Experimental results show that the polymer c-1 can be used for high-efficiency transfection of siRNA, and ROS-triggered gene release plays a key role in improving gene transfection efficiency and cell level selective gene silencing.

Example 19: cell transfection Activity of Polymer d-1/siRNA nanocomposites

Preparing a polymer d-1/siEGFP nano-composite by adopting the polymer d-1 and siRNA (siEGFP) targeting EGFP protein; polymer d-1/siNC nanocomposites were prepared with Polymer d-1 and negative control siRNA (siNC) as controls.

Taking 4T1-GFP cells stably expressing GFP in logarithmic growth phase, collecting cells by trypsinization, and performing 1 × 10 cell selection⁴The cells were plated at a density of one well in 24-well plates and the total volume of the medium was 0.5 mL. 5% CO at 37 ℃₂Incubate overnight. The original medium was discarded and the cells were washed and replaced with serum-free medium containing polymer d-1/siEGFP, polymer d-1/siNC nanocomposite, untreated 4T1-GFP cells were used as a blank control, and in addition, cells were treated with serum-free medium containing 10mM antioxidant N-acetyl-L-cysteine (NAC) and polymer d-1/siEGFP, with siRNA concentrations of each group being 100 nM. Cells were incubated at 37 ℃ with 5% CO₂The culture was continued for 8h in an incubator with replacement of fresh serum-free medium, wherein NAC-treated cells were cultured for 24h with serum-free medium containing 10mM NAC. The expression level of the reporter gene GFP was observed under an inverted fluorescence microscope and a photograph was taken. The experimental results are shown in FIG. 30, compared with untreated 4T1-GFP cells, the polymer d-1/siNC nano-composite does not affect the GFP expression level of 4T1-GFP cells, the polymer d-1/siEGFP obviously inhibits the GFP expression, and the transfection efficiency of the polymer d-1/siEGFP is obviously reduced after intracellular ROS is eliminated by NAC. Experimental results show that the polymer d-1 can be used for high-efficiency transfection of siRNA, and ROS-triggered gene release plays a key role in improving gene transfection efficiency and cell level selective gene silencing.

Example 20: cell transfection Activity of Polymer e-1/siRNA nanocomposites

Preparing a polymer e-1/siEGFP nano-composite by using the polymer e-1 and siRNA (siEGFP) targeting EGFP protein; polymer e-1/siNC nanocomposites were prepared with Polymer e-1 and negative control siRNA (siNC) as controls.

Taking 4T1-GFP cells stably expressing GFP in logarithmic growth phase, collecting cells by trypsinization, and performing 1 × 10 cell selection⁴The cells were plated at a density of one well in 24-well plates and the total volume of the medium was 0.5 mL. 5% CO at 37 ℃₂Incubate overnight. Discard original medium and wash cells, replace with polymer e-1/siEGFP, polymerSerum-free medium of e-1/siNC nanocomplex, untreated 4T1-GFP cells were used as blank control, and in addition, cells were treated with serum-free medium containing 10mM antioxidant N-acetyl-L-cysteine (NAC) and polymer e/siefp, with siRNA concentrations of 100nM for each group. Cells were incubated at 37 ℃ with 5% CO₂The culture was continued for 8h in an incubator with replacement of fresh serum-free medium, wherein NAC-treated cells were cultured for 24h with serum-free medium containing 10mM NAC. The expression level of the reporter gene GFP was observed under an inverted fluorescence microscope and a photograph was taken. The experimental results are shown in FIG. 31, compared with untreated 4T1-GFP cells, the polymer e-1/siNC nano-composite does not affect the GFP expression level of 4T1-GFP cells, the polymer e-1/siEGFP obviously inhibits the GFP expression, and the transfection efficiency of the polymer e-1/siEGFP is obviously reduced after intracellular ROS is eliminated by NAC. Experimental results show that the polymer e-1 can be used for high-efficiency transfection of siRNA, and ROS-triggered gene release plays a key role in improving gene transfection efficiency and cell level selective gene silencing.

The polymers a-2, a-3, b-2, b-3, c-2, c-3, d-2, d-3, e-2, e-3 prepared in examples 1-5 all had the same or similar functions and effects as a-1, b-1, c-1, d-1 and e-1.

Claims

1. A super-charge cationic polymer is characterized in that the super-charge cationic polymer is an aliphatic amine polymer and comprises an active oxygen response group and a quaternary ammonium salt structure; the active oxygen response group is a group capable of being oxidized and hydrolyzed under the action of active oxygen and is selected from one or more of aromatic boric acid, aromatic boric acid ester, oxalic acid monoaromatic ester and oxalic acid diarylester; the quaternary ammonium salt structure has an ultra-high density positive charge.

2. The supercharged cationic polymer of claim 1, wherein the supercharged cationic polymer is selected from any of the structures shown in any one of formulae (i), (ii), and (iii) wherein R is selected from the group consisting of₁、R₂、R₃Independently selected from hydroxyl, alkyl or aromatic hydrocarbon with carbon number not more than 20, or R₁、R₂Passing throughThe chemical bond is connected into a ring, the sum of n or x + y is an integer of 50-1000, the algebra of PAMAM is 2.0-7.0:

3. use of the supercharged cationic polymer of claims 1 or 2 in the preparation of a biomacromolecule drug loading and delivery system.

4. The use according to claim 3, wherein the biomacromolecule drug is selected from the group consisting of nucleic acids, polypeptides, proteinaceous biomacromolecules and derivatives thereof.

5. A transfection reagent, which is characterized in that the transfection reagent comprises the super-charge cationic polymer of any one of claims 1-2 and a transfection reagent working solution, wherein the transfection reagent working solution is selected from one or more of PBS solution, normal saline, 5% glucose solution and cell culture medium.

6. The method for preparing an active oxygen-triggered charge annihilation-type supercharged cationic polymer transfection reagent according to claim 5, characterized by comprising the steps of:

7. The method of claim 6, wherein:

the aliphatic amine polymer in the step (1) is selected from one of polyethyleneimine, polyenepolyamine, polylysine, chitosan and aminoethylethanolamine dendrimer; the reagents used in the reductive amination reaction comprise organic aldehyde and organic carboxylic acid, and the reaction molar ratio of amine groups in the aliphatic amine polymer to aldehyde groups in the organic aldehyde is 1: (1.5-10), wherein the reaction molar ratio of the amino group in the aliphatic amine polymer to the carboxyl group in the organic carboxylic acid is 1: (2.2-10);

8. The preparation method according to claim 6, wherein the reductive amination in the step (1) is carried out at a reaction temperature of 60-100 ℃ for 6-24 hours; the separation and purification method comprises the steps of adding acid liquor into reaction liquid, evaporating the reaction liquid to dryness, dissolving obtained solid in water, adding alkali liquor, separating an organic phase, and drying to obtain the tertiary aminated fatty amine polymer.

9. Use of the transfection reagent of claim 5 for transfection of a biomacromolecule drug selected from the group consisting of nucleic acids, polypeptides and proteinaceous biomacromolecules and derivatives thereof.

10. Use of a transfection reagent according to claim 9, characterized in that it comprises the following steps:

(1) dissolving the biological macromolecular drug in physiological solution, adding a transfection reagent, and uniformly mixing to obtain a super-charge cationic polymer/biological macromolecular drug compound solution;

(2) the solution of the super-charge cationic polymer/biomacromolecule drug compound can be used for in vitro cell transfection, or can be used for one or more of intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, focal local injection, nasal cavity administration, eye administration and lung administration.