阅读说明：本技术 基于树枝状阳离子聚酰胺和四苯乙烯的靶向结肠部位抗菌呈像纳米材料的制备方法 (Preparation method of targeted colon part antibacterial imaging nano material based on dendritic cationic polyamide and tetraphenylethylene ) 是由 高辉 刘宏宇 勾向博 闫香洁 于 2020-10-31 设计创作，主要内容包括：一种基于树枝状阳离子聚酰胺和四苯乙烯的靶向结肠部位抗菌呈像纳米材料的制备方法,属于纳米材料自组装技术领域。所述自组装纳米材料为以三代聚酰胺为基础高分子,以共价键的方式连接偶氮苯基团,进而通过甲基化反应得到季铵盐抗菌材料,利用正负电荷引入羧基化的四苯基乙烯。偶氮苯部分用作结肠靶向位点,并通过共价键与聚酰胺共轭,然后通过甲基化反应形成目标树突状阳离子季铵盐材料,以制备季铵偶氮苯聚酰胺。随后,我们通过静电相互作用引入了四苯乙烯,通过聚集诱导了用于结肠部位可视化的发光效果。本发明简便易行、成本低廉,可广泛应用于材料学、生物学、医学等领域,既可以抑制结肠部位细菌的滋生,又可以作为荧光探针实现可视化检测。(A preparation method of a target colon part antibacterial imaging nano material based on dendritic cationic polyamide and tetraphenylethylene belongs to the technical field of nano material self-assembly. The self-assembly nano material is a polymer based on three-generation polyamide, azobenzene groups are connected in a covalent bond mode, a quaternary ammonium salt antibacterial material is obtained through methylation reaction, and carboxylated tetraphenylethylene is introduced by positive and negative charges. The azobenzene moiety serves as a colon targeting site and is conjugated to the polyamide via a covalent bond, followed by methylation to form the target dendritic cationic quaternary ammonium salt material to produce the quaternary ammonium azobenzene polyamide. Subsequently, we introduced tetraphenylethylene by electrostatic interaction, inducing by aggregation a luminescent effect for visualization of colon sites. The invention is simple and easy to operate, has low cost, can be widely applied to the fields of materials science, biology, medicine and the like, can inhibit the breeding of bacteria at the colon part, and can be used as a fluorescent probe to realize visual detection.)

1. The preparation method of the target colon part antibacterial imaging nano material based on dendritic cationic polyamide and tetraphenylethylene is characterized in that the self-assembled nano material is a polymer based on tertiary Polyamide (PAMAM), azobenzene groups (AZO) are connected in a covalent bond mode, and then a quaternary ammonium salt antibacterial material is obtained through methylation reaction, and carboxylated tetraphenylethylene is introduced by utilizing positive and negative charges, and the preparation method comprises the following steps:

1) adding polyamide amine (PAMAM), 4-methylmorpholine (NMM) and 4- (dimethylamino) azobenzene 4' -isothiocyanate (AZO) into N, N-Dimethylformamide (DMF) dropwise, stirring at room temperature overnight to obtain target high molecular azobenzene polyamide (PAMAM-AZO), dialyzing with ultrapure water, and freeze-drying to obtain a powder product;

2) dissolving the above PAMAM-AZO in H₂Adding excessive methyl iodide dropwise into the O/DMSO mixture, stirring at room temperature overnight, removing excessive methyl iodide from the reaction solution, dialyzing the crude product with sodium chloride solution, dialyzing with distilled water, and lyophilizing to obtain powder product, quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO);

3) dissolving the above Q-PAMAM-AZO in PBS buffer, adding carboxylated tetraphenylethylene [ TPE- (COOH)₄]And (3) performing shake bed incubation at room temperature to obtain the self-assembled nano material (TPE @ Q-PAMAM-AZO).

2. The preparation method of the antibacterial imaging nano material targeting colon parts of dendritic cationic polyamide and tetraphenylethylene according to claim 1, wherein the azobenzene polyamide is dendritic azobenzene polyamide (PAMAM-AZO) modified by dendritic polymer polyamide; the dendrimer polyamide is a 3-generation ethylenediamine core.

3. The preparation method of the antibacterial imaging nano material targeting colon parts of dendritic cationic polyamide and tetraphenylethylene according to claim 1, which is characterized in that the molar ratio of Polyamide (PAMAM) to 4- (dimethylamino) azobenzene 4' -isothiocyanate (AZO) is 6: 1.

4. the preparation method of the antibacterial imaging nano material targeting colon parts of dendritic cationic polyamide and tetraphenylethylene according to claim 1, which is characterized in that in the step 2), PAMAM-AZO is reacted with methyl iodide, and the molar ratio of the PAMAM-AZO to the methyl iodide is 1: and 10, obtaining quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO) after reaction.

5. The preparation method of the antibacterial imaging nano material targeting colon parts of dendritic cationic polyamide and tetraphenylethylene according to claim 1, wherein in the step 3), Q-PAMAM-AZO and carboxylated tetraphenylethylene [ TPE- (COOH) are added₄]Incubate at 37 ℃ at 150 rpm in a shaker.

6. The preparation method of the antibacterial imaging nano material targeting colon parts of the dendritic cationic polyamide and tetraphenylethylene according to claim 1, which is characterized in that H in the step 2)₂The volume ratio of O/DMSO is 1: 1.

Technical Field

The invention belongs to the technical field of nano material self-assembly, and utilizes the action of positive and negative charges. Under the characteristic of hydrophilic self-assembly of quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO), the surface of quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO) is coated with carboxylated tetraphenylethylene [ TPE- (COOH) by utilizing the mutual attraction of positive and negative charges₄]The method for preparing nanoparticles of (1).

Background

Self-assembly, refers to a technique in which basic building blocks (molecules, nanomaterials, substances on the micrometer or larger scale) spontaneously form an ordered structure. During the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds. Self-assembly is based on the properties of the constituent elements (e.g., shape, surface properties, charge, polarizability, magnetic dipole, mass, etc.) that determine their interaction. The design of a system that organizes itself into a desired structure and function is critical to self-assembly applications. The basic structural units must be able to move relative to one another. The balance of attractive and repulsive forces between them determines the steady state position. Molecular self-assembly involves non-covalent or weak covalent interactions (van der waals, electrostatic and hydrophobic interactions, hydrogen and coordination bonds). During self-assembly of macromolecules, interactions of macroscopic objects can often be selected and adjusted, and can include interactions such as attraction, external electromagnetic fields, magnetic fields, capillaries, and entropy, which are not important in the case of molecules. Since self-assembly requires that the components be mobile, it is usually performed on a liquid phase or a smooth surface. The self-assembling environment may regulate the interaction of the self-assembly. And the use of border and other assembly templates is particularly important in self-assembly because the templates can reduce defects and control structures. Equilibrium is generally required to achieve an ordered structure. If the base units are irreversibly stuck together during assembly, they form a glassy state rather than a crystalline or other regular structure. Self-assembly requires that the basic units be balanced between an aggregated state and a non-aggregated state, or that their positions relative to each other be adjusted during aggregation.

The nano particles prepared by the method are characterized in that dendritic polymer polyamide PAMAM is used as a basic framework, azobenzene AZO is connected through a covalent bond to be used as a colon part targeting group, then quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO) which is a quaternary ammonium salt antibacterial material is obtained through methylation reaction, and simultaneously, carboxyl modified tetraphenylethylene derivatives with an aggregation luminescence effect are coated through the action of positive and negative charges, so that the self-assembly nano material (TPE @ Q-PAMAM-AZO) can be obtained.

Disclosure of Invention

The invention aims to provide a preparation method of a self-assembled nano material with bacteriostatic property and targeted imaging aiming at the technical analysis, and the preparation method has the advantages of simple process, low cost and easy implementation.

The technical scheme of the invention is as follows:

the preparation method of the target colon part antibacterial imaging nano material based on dendritic cationic polyamide and tetraphenylethylene is characterized in that the self-assembled nano material is a polymer based on tertiary Polyamide (PAMAM), azobenzene groups (AZO) are connected in a covalent bond mode, a quaternary ammonium salt antibacterial material is further obtained through methylation reaction, and carboxylated tetraphenylethylene is introduced by utilizing positive and negative charges, and the preparation method comprises the following steps:

1) adding Polyamide (PAMAM), 4-methylmorpholine (NMM) and 4- (dimethylamino) azobenzene 4' -isothiocyanate (AZO) into N, N-Dimethylformamide (DMF) dropwise, stirring at room temperature overnight to obtain target high molecular azobenzene polyamide (PAMAM-AZO), dialyzing with ultrapure water, and lyophilizing to obtain powder product;

2) dissolving the PAMAM-AZO in 10mL of H₂O/DMSO (1: 1) mixture, then adding excess methyl iodide dropwise to the solution, stirring at room temperature overnight, removing excess methyl iodide from the reaction solution, and then adding sodium chloride solution to the crude productDialyzed, then dialyzed against distilled water. Finally, freeze-drying to obtain a powder product, namely quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO);

3) dissolving the above Q-PAMAM-AZO in PBS buffer, adding carboxylated tetraphenylethylene [ TPE- (COOH)₄]And (3) performing shake bed incubation at room temperature to obtain the self-assembled supermolecule nano material (TPE @ Q-PAMAM-AZO).

The preparation method of the target colon antibacterial imaging nano material of the dendritic cationic polyamide and tetraphenylethylene is characterized in that the azobenzene polyamide is dendritic azobenzene polyamide (PAMAM-AZO) modified by dendritic polymer polyamide (3-generation ethylenediamine core).

The preparation method of the dendritic cationic polyamide and tetraphenylethylene targeted colon antibacterial imaging nano material is characterized in that the molar ratio of polyamide amine (PAMAM) to 4- (dimethylamino) azobenzene 4' -isothiocyanate (AZO) is 6: 1.

further, in the step 2), PAMAM-AZO is reacted with methyl iodide in a molar ratio of 1: and 10, obtaining quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO) after reaction.

Further, in step 3), Q-PAMAM-AZO and carboxylated tetraphenylethylene [ TPE- (COOH)₄]Incubate at 37 ℃ at 150 rpm in a shaker.

The method has the advantages that the azobenzene part is used as a colon targeting site, is conjugated with polyamide through a covalent bond, and then forms a target dendritic cationic quaternary ammonium salt material through methylation reaction to prepare the quaternary ammonium azobenzene polyamide. Subsequently, we introduced tetraphenylethylene by electrostatic interaction, inducing by aggregation a luminescent effect for visualization of colon sites. The invention is simple and easy to operate, has low cost, can be widely applied to the fields of materials science, biology, medicine and the like, can inhibit the breeding of bacteria at the colon part, and can be used as a fluorescent probe to realize visual detection.

The invention is simple and easy to operate, has low cost, can be widely applied to the fields of materials science, biology, medicine and the like, can inhibit the breeding of bacteria at the colon part, and can be used as a fluorescent probe to realize visual detection.

Drawings

FIG. 1 shows the design and action mechanism of self-assembled nano-particle antibacterial material.

FIG. 2 shows transmission electron micrographs of (ｃA) Q-P-A and (b) TPE @ Q-P-A.

FIG. 3 is an infrared plot of PAMAM-AZO and Q-P-A.

FIG. 4 is ｃA plot of the fluorescence of TPE @ Q-P-A after targeting.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

FIG. 1 shows the design and action mechanism of the self-assembled nanoparticle antibacterial imaging material of the present invention, as can be seen from FIG. 1,

the self-assembly nano material is a self-assembly nano material (TPE Q-PAMAM-AZO) which is obtained by taking three-generation Polyamide (PAMAM) as a basic polymer, connecting azobenzene groups (AZO) in a covalent bond mode, further obtaining a quaternary ammonium salt antibacterial material through methylation reaction and introducing carboxylated tetraphenylethylene by utilizing positive and negative charges.

The following is a description of specific examples of the invention:

polyamide (PAMAM) (14.49nmol) and 4-methylmorpholine (NMM) (5.31mmol), 4- (dimethylamino) azobenzene 4' -isothiocyanate (AZO) (23.17nmol) were added dropwise to N, N-Dimethylformamide (DMF) (10ml) and the resulting mixture was stirred at room temperature overnight. After stirring overnight at room temperature. The crude product was then dialyzed against DMF for 2 days to remove unreacted AZO, and then against distilled water for 2 days. Here, a crude PAMAM-AZO solution was obtained. The crude PAMAM-AZO was then filtered through ｃA 0.22 μm filter, we obtained nanoparticles PAMAM-AZO of appropriate size, and by freeze-drying the final solution, the obtained PAMAM-AZO (P- ｃA) was obtained as an orange powder.

Using Q-P-A as an example, 20mg of PAMAM-AZO was dissolved in 10mL of H₂O/DMSO (volume ratio 1: 1) mixture, then excess methyl iodide (198mg, 1.4mmol) was added dropwise to the solution. After stirring at room temperature overnight, excess methyl iodide was removed from the reaction solution. The crude product is then dialyzed against sodium chloride solution for 2 days and then evaporatedDistilled water was dialyzed for another 2 days. Insoluble material was filtered and the final solution was freeze dried to give the resulting quaternary ammonium azobenzene polyamide Q-P-A as an orange powder.

To process a semiconductor wafer with a 1: 4 TPE: molar ratio of Q-P- ｃA supramolecular nanoparticles TPE @ Q-P- ｃA were obtained and ｃA 0.5mg/mL PBS solution containing TPE (pH 7.4) was prepared. Dissolving the above Q-P-A in PBS buffer, adding carboxylated tetraphenylethylene [ TPE- (COOH)₄]And (3) carrying out shake bed incubation at room temperature, and carrying out shake bed incubation for 30 minutes at the rotation speed of 150 and the temperature of 37 ℃ to obtain the self-assembled nano material (TPE @ Q-PAMAM-AZO).

FIG. 2 shows transmission electron micrographs of (ｃA) Q-P-A and (b) TPE @ Q-P-A according to the present invention.

The figure shows that the diameter of Q-P-A is 184 + -10 nm and is spherical as seen in ｃA transmission electron micrograph (FIG. 2 ｃA). And the surface can be seen to be smooth. The TPE @ Q-P-A obtained shows ｃA significant change in transmission electron microscopy after attraction of TPE by positive and negative charges, the change in morphology is attributed to the adsorption of TPE on the surface of Q-P-A (FIG. 2 b).

FIG. 3FT-IR Spectrum 1250cm^-1the-N-H-stretching of the peak confirms the aminolysis of the terminal amino group of PAMAM, confirms the occurrence of quaternization reaction and confirms the successful construction of the antibacterial material quaternary ammonium azobenzene polyamide (Q-PAMAM-AZO).

FIG. 4 shows fluorescence intensity (excitation wavelength is 400nm) measured after ｃA nanometer antibacterial probe TPE @ Q-P-A is added with ｃA reducing agent. The fluorescence emission intensity of the destroyed TPE @ Q-P-A increases significantly around 500nm after treatment with the reducing agent (FIG. 4). After the azo is destroyed, a change in the state of the particles is induced, and the AIE effect of the TPE is activated and re-aggregates to produce strong fluorescence.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are only used for describing the present invention

Further description, the scope of the present invention is not limited thereby. The technical idea of the invention is that only obvious changes are needed and still fall within the scope of the invention.