阅读说明：本技术 具有氧化应激损伤修复的基因洗脱涂层材料及其制备方法 (Gene elution coating material with oxidative stress damage repair function and preparation method thereof ) 是由 余泓池 刘肖珩 向茂龙 马佳 罗日方 曹成建 沈阳 于 2021-01-28 设计创作，主要内容包括：本发明提供了一种具有氧化应激损伤修复的基因洗脱涂层材料及其制备方法,属于生物医学工程功能材料技术。其制备方法包括：对经清洗后的金属基底材料进行表面氨基官能化处理。将上述材料置于pH为3～5的富含强氧化剂的溶液中,加入浓度为0.1～5mg/mL的亲水性化合物溶液,获得具有微纳拓扑结构,同时富含反应性醌基和氨基的目标材料A。制备可增强表达Tom70的慢病毒基因载体目标材料B,其中,载体为慢病毒载体。将目标材料B固定在目标材料A表面,即获得目标具有氧化应激损伤修复的基因洗脱涂层材料。该涂层具有微纳拓扑结构,可为基因载体提供保护性的温床,为基因递送创造有利条件。(The invention provides a gene eluting coating material with oxidative stress damage repair function and a preparation method thereof, belonging to the biomedical engineering functional material technology. The preparation method comprises the following steps: and carrying out surface amino functionalization treatment on the cleaned metal substrate material. The material is placed in a strong oxidant-rich solution with the pH value of 3-5, and a hydrophilic compound solution with the concentration of 0.1-5 mg/mL is added to obtain a target material A which has a micro-nano topological structure and is rich in reactive quinonyl and amino. Preparing a target material B of a lentiviral vector capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector. And (3) fixing the target material B on the surface of the target material A to obtain the target gene eluting coating material with the function of repairing oxidative stress damage. The coating has a micro-nano topological structure, can provide a protective hotbed for a gene vector, and creates favorable conditions for gene delivery.)

1. A preparation method of a gene-eluting coating material with oxidative stress damage repair is characterized by comprising the following steps:

(1) carrying out surface amino functionalization treatment on the cleaned metal substrate material;

(2) placing the material obtained in the step (1) in a strong oxidant-rich solution with the pH of 3-5, adding a hydrophilic compound solution with the concentration of 0.1-5 mg/mL, controlling the reaction temperature to be 55-65 ℃, further reacting for 5-120 min, cleaning for 1-3 times, and drying with nitrogen to obtain a target material A which has a micro-nano topological structure and is rich in reactive quinonyl and amino; wherein the hydrophilic compound is a mixture of tannic acid and polyethyleneimine;

(3) preparing a lentiviral vector target material B capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector;

(4) and fixing the target material B on the surface of the target material A to obtain the gene eluting coating material with the function of repairing oxidative stress damage.

2. The method for preparing a gene eluting coating material with oxidative stress damage repair function as claimed in claim 1, wherein the metal substrate material is selected from one of iron alloy, magnesium alloy, zinc alloy, titanium alloy and cobalt-chromium alloy.

3. The method for preparing a gene-eluting coating material with oxidative stress damage repair function according to claim 1, wherein the surface amino-functionalization treatment is one selected from an amino plasma treatment, a dopamine solution soaking treatment and a mixed solution soaking treatment of dopamine and polylysine.

4. The method for preparing a gene-eluting coating material with oxidative stress damage repair according to claim 1, wherein the strong oxidant is selected from one of persulfate, copper sulfate, periodate and fenton's reagent.

5. The method for preparing a gene eluting coating material with oxidative stress damage repair function according to claim 1, wherein the method for fixing the target material B on the target material A is a covalent bonding method.

6. The method for preparing a gene eluting coating material with oxidative stress damage repair function according to claim 5, wherein the covalent bonding method is a Michael addition reaction formed between an amino group on the target material B and a quinone group on the target material A.

7. The method for preparing a gene eluting coating material with oxidative stress damage repair function according to claim 6, wherein the Michael addition reaction is performed in a solution system with pH of 7.5-9.

8. The method for preparing a gene eluting coating material with oxidative stress damage repair function according to claim 5, wherein the covalent bonding method is an amide bond formed by dehydration condensation between a carboxyl group on the target material B and an amino group on the target material A.

9. The method for preparing a gene eluting coating material with oxidative stress damage repair function according to claim 8, wherein the amide bond formed by dehydration condensation is performed in a solution system with a reaction system pH of 5-7.

10. A gene-eluting coating material having oxidative stress damage repair properties, which is prepared by the preparation method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of biomedical engineering functional materials, and particularly relates to a gene elution coating material with oxidative stress damage repair function and a preparation method thereof.

Background

Vascular Stent implantation has become the main means for treating coronary heart disease, however, the early Metal Bare Stent (BMS) is easy to cause excessive hyperplasia of neointima after being implanted, thereby causing high incidence (20-30%) of In-Stent Restenosis (ISR); while Drug-eluting Stent (DES) can effectively inhibit the probability of ISR occurrence by in situ release of antiproliferative drugs (such as rapamycin, everolimus, etc.), the following clinical reports show that DES inhibits the vascular smooth muscle cell hyperproliferation and also inhibits the healing delay of endothelial cell layer, resulting in Late thrombus (LST) (incidence rate is about 1.5% -2%), which is a serious hidden danger in DES use. Through systemic clinical analysis, drug efficacy termination, lack of support for endothelial functional recovery, and unresolved inflammatory responses and excessive oxidative stress are the major causes of the long-term failure of drug-coated stents. Among them, inhibiting excessive oxidative stress after stent implantation has become an important means for solving late thrombosis in recent years.

Due to the specificity of Gene therapy in targeting and persistence, Gene-eluting stents (GES) have come into play, and the core is to select appropriate genes for targeted delivery, transfection and expression, thereby playing a role in regulating the repair process of vascular tissues. The mitochondrial outer membrane complex receptor Tom70 plays a key role in regulating intracellular oxidative stress. Overexpression of Tom70 in cells can significantly down-regulate the oxidative stress in cells. However, in the field of cardiovascular scaffolds, no attempt has been made to introduce Tom70 into recipient cells to reduce intracellular oxidative stress.

Disclosure of Invention

The invention provides a gene elution coating material with oxidative stress damage repair function and a preparation method thereof, which can be used for introducing therapeutic genes into receptor cells by utilizing a material coating with a micro-nano topological structure, can reduce oxidative stress reaction after stent implantation, and is expected to solve the problem of late thrombosis.

In a first aspect, the present invention provides a method for preparing a gene-eluting coating material with oxidative stress damage repair, comprising the steps of:

(1) and carrying out surface amino functionalization treatment on the cleaned metal substrate material.

(2) Placing the material obtained in the step (1) in a strong oxidant-rich solution with the pH of 3-5, adding a hydrophilic compound solution with the concentration of 0.1-5 mg/mL, controlling the reaction temperature to be 55-65 ℃, further reacting for 5-120 min, cleaning for 1-3 times, and drying with nitrogen to obtain a target material A which has a micro-nano topological structure and is rich in reactive quinonyl and amino; wherein the hydrophilic compound is a mixture of tannic acid and polyethyleneimine.

(3) Preparing a target material B of a lentiviral vector capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector.

(4) And (3) fixing the target material B on the surface of the target material A to obtain the target gene eluting coating material with the function of repairing oxidative stress damage.

Further, the metal base material is selected from one of iron alloy, magnesium alloy, zinc alloy, titanium alloy and cobalt-chromium alloy.

Further, the surface amino-functionalization treatment mode is selected from one of amino plasma treatment, dopamine solution soaking treatment and mixed solution soaking treatment of dopamine and polylysine.

Further, the strong oxidant is selected from one of persulfate, copper sulfate, periodate and fenton reagent.

Further, the method of immobilizing the target material B on the target material a is a covalent bonding method.

Further, the covalent bonding method used is a Michael addition reaction formed between an amino group on the target material B (amino group on the lentiviral vector) and a quinone group on the target material A.

Further, the Michael addition reaction is carried out in a solution system with the pH value of 7.5-9.

Further, the covalent bonding method used is an amide bond formed by dehydration condensation between a carboxyl group on the target material B (carboxyl group on the lentiviral vector) and an amino group on the target material a.

Further, the amide bond formed by dehydration condensation is carried out in a solution system with the pH value of 5-7.

In a second aspect, the invention provides a gene eluting coating material with oxidative stress damage repair function, which is prepared by the preparation method.

The gene eluting coating material with oxidative stress damage repair and the preparation method thereof provided by the invention have the beneficial effects that:

the hydrophilic target material A has a micro-nano topological structure, is rich in reactive quinonyl and amino on the surface and is fixed with a target material B rich in carboxyl and amino on the surface, and the target material A can provide a fixed foundation for the target material B, so that therapeutic genes of the target material B are introduced into receptor cells through the target material A, the oxidative stress reaction after the stent is implanted can be reduced, and the problem of late thrombosis is expected to be solved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments are briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive efforts and also belong to the protection scope of the present application.

FIG. 1 is an XPS analysis of target material A;

FIG. 2 is a graph showing the evaluation of hydrophilicity and hydrophobicity of the coating provided in example 5;

fig. 3 is a micro-nano topology structure diagram of the coating provided in example 5;

FIG. 4 is a graph of the evaluation of the vascular endothelial cell activity of the coating provided in example 5;

FIG. 5 is a graph showing the expression of Tom70 in vascular endothelial cells of the coating provided in example 5;

FIG. 6 is a water and affinity evaluation chart of the coating provided in comparative example 1;

FIG. 7 is a graph showing the evaluation of the hydrophilicity and hydrophobicity of the coating provided in comparative example 2;

fig. 8 is a water and affinity evaluation chart of the coating provided in comparative example 3.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.

Example 1

A preparation method of a gene-eluting coating material with oxidative stress damage repair comprises the following steps:

(1) mixing a dopamine solution of 1mg/mL and a polyethyleneimine solution of 15mg/mL to obtain a mixed solution, soaking the cleaned iron alloy substrate material in the mixed solution, and carrying out copolymerization for 2h under the condition of a solution with the pH value of 9 to obtain an amino-functionalized surface;

(2) putting the material obtained in the step (1) into a persulfate solution with the pH value of 3, adding a mixture of tannic acid and polyethyleneimine with the concentration of 0.5mg/mL (the concentration of tannic acid is 0.2mg/mL, and the concentration of polyethyleneimine is 0.3mg/mL), controlling the reaction temperature to be 60 ℃, further reacting for 50min, cleaning for 3 times, and then drying with nitrogen to obtain a target material A;

(3) preparing plasmid DNA capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector, and the obtained gene vector is a target material B; the core plasmid is pLenti-EF1 alpha-Tom 70-Flag-6His-CMV-GFP-P2A-Puro, the packaging plasmid is pAXPS and pMD2.G, the plasmids transfect HEK293T cells in a ratio of 4:3:1, cell supernatants are respectively collected after 48h/72h, the cells are filtered by a 0.45 mu m filter, the virus is precipitated by polyethylene glycol (PEG8000) solution with molecular weight of 8000 overnight, and the virus is centrifuged by 4000g to obtain concentrated virus.

(4) Soaking the target material A in water solution with pH of 8.5, and slowly adding appropriate amount of target material B (2 μ g/cm)²) The target material B is fixed on the surface of the target material A.

Example 2

A preparation method of a gene-eluting coating material with oxidative stress damage repair comprises the following steps:

(1) placing the cleaned magnesium alloy substrate material in a low-temperature plasma generator, introducing an allylamine monomer under the vacuum degree of 0.5Pa, and performing glow discharge for 10min to obtain an amino positive charge surface;

(2) placing the material obtained in the step (1) in a copper sulfate solution with pH of 3, adding a mixture of tannic acid and polyethyleneimine (the concentration of tannic acid is 0.2mg/mL, and the concentration of polyethyleneimine is 0.3mg/mL) with the concentration of 0.5mg/mL, controlling the reaction temperature to be 60 ℃, further reacting for 50min, cleaning for 3 times, and then drying with nitrogen to obtain a target material A;

(3) preparing plasmid DNA capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector, and the obtained gene vector is a target material B; the core plasmid is pLenti-EF1 alpha-Tom 70-Flag-6His-CMV-GFP-P2A-Puro, the packaging plasmid is pAXPS and pMD2.G, the plasmids transfect HEK293T cells in a ratio of 4:3:1, cell supernatants are respectively collected after 48h/72h, the cells are filtered by a 0.45 mu m filter, the virus is precipitated by polyethylene glycol (PEG8000) solution with molecular weight of 8000 overnight, and the virus is centrifuged by 4000g to obtain concentrated virus.

(4) Soaking the target material A in water solution with pH of 8.5, and slowly adding appropriate amount of target material B (2 μ g/cm)²) The target material B is fixed on the surface of the target material A.

Example 3

A preparation method of a gene-eluting coating material with oxidative stress damage repair comprises the following steps:

(1) placing the cleaned zinc alloy substrate material in a low-temperature plasma generator, introducing an allylamine monomer under the vacuum degree of 0.5Pa, and performing glow discharge for 10min to obtain an amino positive charge surface;

(2) placing the material obtained in the step (1) in a periodate solution with pH of 3, adding a mixture of tannic acid and polyethyleneimine with the concentration of 2mg/mL (the concentration of tannic acid is 0.5mg/mL, and the concentration of polyethyleneimine is 1.5mg/mL), controlling the reaction temperature to be 60 ℃, further reacting for 90min, cleaning for 3 times, and then drying with nitrogen to obtain a target material A;

(3) preparing plasmid DNA capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector, and the obtained gene vector is a target material B; the core plasmid is pLenti-EF1 alpha-Tom 70-Flag-6His-CMV-GFP-P2A-Puro, the packaging plasmid is pAXPS and pMD2.G, the plasmids transfect HEK293T cells in a ratio of 4:3:1, cell supernatants are respectively collected after 48h/72h, the cells are filtered by a 0.45 mu m filter, the virus is precipitated by polyethylene glycol (PEG8000) solution with molecular weight of 8000 overnight, and the virus is centrifuged by 4000g to obtain concentrated virus.

(4) Soaking the target material A in water solution with pH of 8.5, and slowly adding appropriate amount of target material B (2 μ g/cm)²) The target material B is fixed on the surface of the target material A.

Example 4

A preparation method of a gene-eluting coating material with oxidative stress damage repair comprises the following steps:

(1) mixing a dopamine solution of 1mg/mL and a polyethyleneimine solution of 15mg/mL to obtain a mixed solution, soaking the cleaned titanium alloy substrate material in the mixed solution, and carrying out copolymerization for 2h under the condition of a solution with the pH of 8 to obtain an amino-functionalized surface;

(2) placing the material obtained in the step (1) in a periodate solution with pH of 3, adding a mixture of tannic acid and polyethyleneimine with the concentration of 3mg/mL (the concentration of tannic acid is 1mg/mL, and the concentration of polyethyleneimine is 2mg/mL), controlling the reaction temperature to be 60 ℃, further reacting for 120min, cleaning for 3 times, and drying with nitrogen to obtain a target material A;

(3) preparing plasmid DNA capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector, and the obtained gene vector is a target material B; the core plasmid is pLenti-EF1 alpha-Tom 70-Flag-6His-CMV-GFP-P2A-Puro, the packaging plasmid is pAXPS and pMD2.G, the plasmids transfect HEK293T cells in a ratio of 4:3:1, cell supernatants are respectively collected after 48h/72h, the cells are filtered by a 0.45 mu m filter, the virus is precipitated by polyethylene glycol (PEG8000) solution with molecular weight of 8000 overnight, and the virus is centrifuged by 4000g to obtain concentrated virus.

(4) Soaking the target material A in water solution with pH of 8.5, and slowly adding appropriate amount of target material B (2 μ g/cm)²) The target material B is fixed on the surface of the target material A.

Example 5

A preparation method of a gene-eluting coating material with oxidative stress damage repair comprises the following steps:

(1) carrying out surface amino functionalization treatment on the cleaned cobalt-chromium alloy substrate material; copolymerizing 0.5mg/mL tannic acid and 2mg/mL lysine for 3h in a solution having a pH of 9 to obtain an amino-rich surface;

(2) placing the material obtained in the step (1) in a Fenton reagent solution with pH of 3, adding a mixture of tannic acid and polyethyleneimine with the concentration of 5mg/mL (the concentration of tannic acid is 1mg/mL, and the concentration of polyethyleneimine is 4mg/mL), controlling the reaction temperature to be 60 ℃, further reacting for 120min, cleaning for 3 times, and drying with nitrogen to obtain a target material A;

(3) preparing plasmid DNA capable of enhancing Tom70 expression, wherein the vector is a lentiviral vector, and the obtained gene vector is a target material B; the core plasmid is pLenti-EF1 alpha-Tom 70-Flag-6His-CMV-GFP-P2A-Puro, the packaging plasmid is pAXPS and pMD2.G, the plasmids transfect HEK293T cells in a ratio of 4:3:1, cell supernatants are respectively collected after 48h/72h, the cells are filtered by a 0.45 mu m filter, the virus is precipitated by polyethylene glycol (PEG8000) solution with molecular weight of 8000 overnight, and the virus is centrifuged by 4000g to obtain concentrated virus.

(4) Soaking the target material A in water solution with pH of 8.5, and slowly adding appropriate amount of target material B (2 μ g/cm)²) The target material B is fixed on the surface of the target material A.

Comparative example 1

A surface amination treatment method of a metal substrate comprises the following steps:

(1) carrying out surface amino functionalization treatment on the cleaned cobalt-chromium alloy substrate material; amino-rich surfaces were obtained by copolymerization of 0.5mg/mL tannic acid and 2mg/mL lysine for 3h in a solution at pH 9.

Comparative example 2

A method for preparing a target material A comprises the following steps:

(1) carrying out surface amino functionalization treatment on the cleaned cobalt-chromium alloy substrate material; amino-rich surfaces were obtained by copolymerization of 0.5mg/mL tannic acid and 2mg/mL lysine for 3h in a solution at pH 9.

(2) And (2) placing the material obtained in the step (1) in a Fenton reagent solution with the pH value of 3, adding tannic acid with the concentration of 5mg/mL, controlling the reaction temperature to be 60 ℃, further reacting for 120min, cleaning for 3 times, and drying with nitrogen to obtain a target material A.

Comparative example 3

A method for preparing a target material A comprises the following steps:

(1) carrying out surface amino functionalization treatment on the cleaned cobalt-chromium alloy substrate material; amino-rich surfaces were obtained by copolymerization of 0.5mg/mL tannic acid and 2mg/mL lysine for 3h in a solution at pH 9.

(2) And (2) placing the material obtained in the step (1) in a Fenton reagent solution with the pH value of 3, adding a mixture of polyethyleneimine with the concentration of 5mg/mL, controlling the reaction temperature to be 60 ℃, further reacting for 120min, cleaning for 3 times, and drying with nitrogen to obtain a target material A.

Test example 1

As can be seen from fig. 1 (high resolution spectrogram of C, N, O), the surface of the target material a contains various groups such as amino groups, phenolic hydroxyl groups, quinone groups, etc., and can be combined with the target material B in different reaction modes).

Planting vascular endothelial cells (1X 10)⁷One) of the coatings obtained in example 5, vascular endothelial cells were collected after 48h and examined for their activity and expression of Tom 70. As shown in fig. 2, the coating hasGood hydrophilic property, and the coating surface has a remarkable wiener topological structure, as shown in figure 3. After the vascular endothelial cells are adhered, the endothelial cells have good cell activity (normal cultured endothelial cells are taken as a control), and as shown in figure 4, the endothelial cells containing the coating have higher proliferation capacity than the normal cultured endothelial cells. In addition, after 2 days of gene delivery, the cells on the surface of the coating were collected and lysed to obtain a protein solution, and the expression of Tom70 in endothelial cells was analyzed using western-blot. Tom70 showed high expression in vascular endothelium, as shown in FIG. 5, indicating that the coating can effectively deliver the gene of interest into cells and achieve intracellular expression.

As can be seen from fig. 6, if the alloy substrate is only aminated and not added with tannic acid and polyethyleneimine for hydrophilic treatment, the hydrophilic effect of the obtained target material is not good, which is not beneficial to the preparation of the subsequent coating.

As can be seen from fig. 7 and 8, if the alloy substrate material is aminated and then treated with tannic acid alone or polyethyleneimine alone for hydrophilic treatment, the hydrophilic effect is inferior to that of the hydrophilic treatment in example 5, which indicates that tannic acid and polyethyleneimine have a certain synergistic effect, and the combination of tannic acid and polyethyleneimine can optimize the hydrophilic effect of the target material a, so that the subsequent coating preparation can improve the coating effect.