Bionic high-biological-activity antioxidant defense liposome system, application and construction method

Abstract

The invention belongs to the technical field of anti-oxidation systems, and discloses a bionic high-biological-activity anti-oxidation defense lipidosome system, application and a construction method.The bionic high-biological-activity anti-oxidation defense lipidosome system is characterized in that bionic means is adopted, and elements of an anti-oxidation defense system in biological cells, such as simulated antioxidant enzymes, water-soluble antioxidant factors and fat-soluble antioxidant factors, are combined into a whole in a bionic manner; layered entrapment is carried out in the form of liposome vesicles, and the center of the system is a water-soluble antioxidant factor; the outer side of the water-soluble antioxidant factor is coated with a lipidosome double-layer structure, and the lipidosome double-layer structure is embedded with the fat-soluble antioxidant factor; a combination of chitosan and Pt nanoclusters is arranged on the outer side of the liposome double-layer structure; the system is applied to preparation of smearing type medicines for resisting oxidative stress inflammation or medicines for improving systemic oxidative stress inflammation diseases. The system constructed by the invention has synergistic oxidation resistance, can effectively remove various active oxygen and active nitrogen, can be used for protecting cell components from oxidative damage, and can promote wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of antioxidant systems, and in particular relates to a biomimetic high-bioactivity antioxidant defense liposome system, its application and construction method. Background Art

[0002] The antioxidant defense system in cells is a molecular-level body defense system established by organisms to maintain the balance between the elimination and generation of free radicals.

[0003] According to the defense order, it is divided into primary antioxidant defense system and antioxidant repair system.

[0004] The primary antioxidant defense system includes antioxidant enzymes, fat-soluble antioxidants, water-soluble antioxidants and protein antioxidants; the antioxidant repair system includes protein repair and degradation, DNA repair system, etc.

[0005] When excessive free radicals are produced in cells, oxidative stress occurs, overwhelms normal antioxidant capacity, and plays a significant role in many disease states. In this situation, traditional antioxidant solutions, which use single-ingredient antioxidants or antioxidant enzymes (such as glutathione and superoxide dismutase) to treat oxidative stress diseases (such as inflammation), suffer from short half-lives, single targets, and poor effectiveness.

[0006] Therefore, a biomimetic high-bioactivity antioxidant defense liposome system, application and construction method are provided. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a biomimetic high-bioactive antioxidant defense liposome system, application and construction method for treating inflammation and aging-related oxidative stress-related diseases.

[0008] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0009] In a first aspect, the present invention provides a biomimetic high bioactive antioxidant defense liposome system, the core of which is a water-soluble antioxidant factor;

[0010] The water-soluble antioxidant factor is wrapped around a liposome bilayer structure, and the liposome bilayer structure contains the fat-soluble antioxidant factor;

[0011] The outer side of the liposome double-layer structure is a combination of chitosan and Pt nanoclusters.

[0012] In some optional examples, the water-soluble antioxidant factor is glutathione (GSH), ascorbic acid or polyphenol, and the fat-soluble antioxidant factor is vitamin E (VE) or coenzyme Q10.

[0013] The present invention constructs an antioxidant defense liposome system through biomimetic means. Pt nanoclusters have excellent superoxidase (SOD) and catalase (CAT)-like abilities under different conditions. Pt nanoclusters combined with glutathione (GSH) and vitamin E (VE) can simulate the primary antioxidant defense system in biological cells and effectively remove various reactive oxygen species and reactive nitrogen species. The system has synergistic antioxidant properties and can effectively remove various reactive oxygen species and reactive nitrogen species. It can be used to protect cell components from oxidative damage and promote wound healing. This system has great potential in treating various diseases caused by oxidative stress.

[0014] In this system, the English abbreviations of each component are as follows:

[0015]

[0016] In a second aspect, the present invention provides an application of a biomimetic high-bioactivity antioxidant defense liposome system as described in the first aspect, which is used to prepare a topical drug for anti-oxidative stress inflammation, or to prepare an oral therapeutic drug for improving systemic oxidative stress inflammatory diseases.

[0017] In a third aspect, the present invention provides a method for constructing a biomimetic high bioactivity antioxidant defense liposome system as described in the first aspect, comprising:

[0018] (I) Preparation of chitosan and Pt nanoclusters;

[0019] (II) preparing inclusion bodies of water-soluble antioxidant factors, fat-soluble antioxidant factors and liposomes;

[0020] (III) adding the inclusion body into the combination body and stirring the mixture thoroughly to obtain a biomimetic high bioactive antioxidant defense liposome system.

[0021] As a preferred technical solution of the present invention, in step (I), the specific steps of preparing the chitosan and Pt nanocluster combination are:

[0022] adding deionized water to chitosan and adjusting the pH to 7.0 to obtain a chitosan solution;

[0023] adding deionized water to a soluble Pt salt to obtain a Pt salt solution;

[0024] The Pt salt solution and the chitosan solution are quickly mixed and stirred to obtain a mixed solution.

[0025] A reducing agent is added to the mixed solution, the pH is adjusted to 8-12, and stirring is continued for 12-72 hours. The solution after stirring is dialyzed and freeze-dried.

[0026] In some optional embodiments, the soluble Pt salt is H2PtCl6 and the reducing agent is NaBH4.

[0027] As a preferred technical solution of the present invention, in step (II), the water-soluble antioxidant factor is glutathione (GSH), and the fat-soluble antioxidant factor is vitamin E (VE). The specific steps of preparing glutathione, vitamin E and liposome inclusion bodies are as follows: dissolving egg yolk lecithin, cholesterol and vitamin E, and removing the solvent by rotary evaporation;

[0028] Add glutathione aqueous solution and hydrate at 37-53°C;

[0029] The liposomes are extruded using a liposome extruder, and the liposomes are repeatedly passed through a 0.05-0.5 μm filter membrane to obtain inclusions of glutathione and liposomes.

[0030] As a preferred technical solution of the present invention, in step (I), the raw materials for preparing the combination of chitosan and Pt nanoclusters include chitosan and soluble Pt salt; in step (II), the water-soluble antioxidant factor is glutathione (GSH), the fat-soluble antioxidant factor is vitamin E (VE), and the raw materials for preparing the inclusion of glutathione, vitamin E and liposomes include egg yolk lecithin, cholesterol, glutathione and vitamin E;

[0031] The mass ratio of glutathione to soluble Pt salt is any ratio in the range of 2:1 to 8:1;

[0032] The mass ratio of egg yolk lecithin to soluble Pt salt is any ratio in the range of 8:1 to 16:1;

[0033] The mass ratio of egg yolk lecithin to cholesterol is any ratio in the range of 1:1 to 8:1;

[0034] The mass ratio of egg yolk lecithin to vitamin E is any ratio in the range of 5:1 to 20:1.

[0035] In some optional examples, the mass ratio of egg yolk lecithin to cholesterol is 4:1.

[0036] The beneficial effects of the present invention are as follows: an antioxidant defense liposome system is constructed by biomimetic means, which has synergistic antioxidant properties, can effectively remove a variety of reactive oxygen and reactive nitrogen species, can be used to protect cell components from oxidative damage, and can promote wound healing. This system has great potential in treating various diseases caused by oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention can be further illustrated by the non-limiting examples given in the accompanying drawings;

[0038] Figure 1This is a schematic diagram of the biomimetic high bioactivity antioxidant defense liposome system constructed in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention are described in detail below in conjunction with specific embodiments and their accompanying drawings. The embodiments described herein are specific embodiments of the present invention and are used to illustrate the concept of the present invention; these descriptions are explanatory and exemplary and should not be understood as limiting the embodiments of the present invention and the scope of protection of the present invention. In addition to the embodiments described herein, those skilled in the art can also adopt other obvious technical solutions based on the contents disclosed in the claims of this application and its specification, including technical solutions that adopt any obvious replacements and modifications to the embodiments described herein.

[0040] Example

[0041] This embodiment is constructed as Figure 1 The biomimetic high bioactivity antioxidant defense liposome system shown comprises the following steps:

[0042] (1) Preparation of a combination of chitosan and Pt nanoclusters

[0043] Take 6 g of chitosan, accurately weigh it, place it in a conical flask, add 450 mL of deionized water, add 5 mL of HCl solution (1 mol / L), and adjust the pH to 7.0 with NaOH solution (0.5 mol / L);

[0044] Take 1 gram of H2PtCl6, accurately weigh it, add 30 mL of deionized water, quickly mix the obtained Pt salt and chitosan solution, stir and add it to a conical flask, take another 0.3 gram of NaBH4, accurately weigh it, place it in a 50 mL volumetric flask, shake it well, add the resulting solution to the conical flask under rapid stirring, slowly adjust the pH to 10.0, continue stirring at room temperature for 12 hours, during which time the pH is always maintained at 10.0 (HCl / NaOH adjustment), place the resulting solution in a dialysis bag (Mw = 3.5 kDa), dialyze it against deionized water (pH 7.0) for 48 hours, and freeze-dry;

[0045] The combination of chitosan and Pt nanoclusters was obtained. Ultra-high resolution transmission electron microscopy showed that the diameter of the Pt nanoclusters was about 1 nm.

[0046] (2) Preparation of glutathione, vitamin E and liposome inclusions

[0047] Take appropriate amounts of egg yolk lecithin, cholesterol, and vitamin E, accurately weigh them, and place them in a 1000 mL eggplant-shaped flask;

[0048] Add 150 mL of solvent to dissolve it, remove the solvent by rotary evaporation, and wait until a uniform film forms on the wall of the bottle. Add 200 mL of preheated glutathione aqueous solution and hydrate at 40°C for 1 hour.

[0049] Use a liposome extruder to extrude 20 times, and make the liposomes pass through a 0.1 μm filter membrane repeatedly to obtain glutathione, vitamin E and liposome inclusions;

[0050] (3) Preparation of biomimetic high bioactive antioxidant defense liposome system

[0051] The inclusion suspension was added dropwise to 2 times the volume of 1% conjugate and stirred at 300 r / min for 1 hour to obtain a biomimetic high bioactivity antioxidant defense liposome system, which was abbreviated as GSH@Pt-CS-VE-LP.

[0052] Efficacy testing

[0053] HaCaT cells were irradiated with ultraviolet B (UVB) at a dose of 40 mJ / cm 2 At this time, the cell survival rate was 63.5%, the density decreased, and the shape became round. The cellular ROS level was observed using an inverted fluorescence microscope. Each treatment group could significantly reduce the ROS level. At a low concentration of 300nM, GSH@Pt-CS-VE-LP reduced the intracellular ROS index by 87%, which was significantly higher than the control group with the same concentration of peroxidase (SOD) and glutathione (GSH) added alone.

[0054] In vivo, the white blood cell indicators in the blood of mice with endotoxemia and systemic inflammation returned to normal after only 3 days, while the white blood cell indicators in the blood of the control group of mice that only added the same concentration of superoxidase (SOD) and glutathione (GSH) remained almost unchanged.

[0055] Stability testing

[0056] The GSH@Pt-CS-VE-LP concentration was adjusted to a low 300nM, and 1% p-hydroxyacetophenone and 1% 1,2-hexanediol were added as the solution preservative system. After being placed at room temperature for 6 months, the solution remained clear and free of precipitation. The nanoparticle morphology under transmission electron microscopy was intact without deformation or breakage. The reduced ROS level in the cells was almost the same as when it was just synthesized. In the control group with the same concentration of peroxidase (SOD), vitamin E (VE) and glutathione (GSH), the solution had already undergone slight precipitation and turbidity and had obviously changed color to yellow.

[0057] Safety Assessment

[0058] Using 2% xanthan gum as the matrix, the effect of nanoliposomes on the viability of skin HaCaT cells was measured at a concentration gradient of 100nM-1000nM GSH@Pt-CS-VE-LP. The experiment showed that at different concentrations, GSH@Pt-CS-VE-LP not only did not reduce cell viability, but also had the effect of enhancing cell viability. Moreover, as the concentration increased from 100nM-1000nM, cell viability increased from 103% to 128%. In the skin application experiment, high concentrations of 10uM-100uM GSH@Pt-CS-VE-LP were continuously used 3 times a day on the exposed skin on the back of mice. There was no irritation on the skin surface. Tissue sections were taken after one week, and the tissue sections were no different from the control group.

[0059] The biomimetic high-bioactive antioxidant defense liposome system proposed in the present invention is also suitable for encapsulating other water-soluble antioxidant factors, such as ascorbic acid, polyphenols, etc., and is also suitable for encapsulating other fat-soluble antioxidant factors, such as coenzyme Q10, etc. Therefore, simple replacement of raw materials is also within the scope of protection of this patent.

[0060] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person skilled in the art without departing from the spirit and technical concepts disclosed herein shall be covered by the claims of the present invention.

Claims

1. A biomimetic high bioactivity antioxidant defense liposome system, characterized by: The center of this system is water-soluble antioxidant factors; The water-soluble antioxidant factor is wrapped around a liposome bilayer structure, and the liposome bilayer structure contains the fat-soluble antioxidant factor; The outer side of the liposome double-layer structure is a combination of chitosan and Pt nanoclusters.

2. The biomimetic high bioactivity antioxidant defense liposome system according to claim 1, characterized in that: The water-soluble antioxidant factor is selected from glutathione, ascorbic acid or polyphenols, and the fat-soluble antioxidant factor is selected from vitamin E or coenzyme Q10.

3. Application of a biomimetic high bioactivity antioxidant defense liposome system, characterized by: The biomimetic high bioactivity antioxidant defense liposome system according to claim 1 is used to prepare a topical drug for anti-oxidative stress inflammation, or to prepare an oral therapeutic drug for improving systemic oxidative stress inflammatory diseases.

4. A method for constructing a biomimetic high-bioactivity antioxidant defense liposome system, characterized by: For constructing the biomimetic high bioactivity antioxidant defense liposome system according to claim 1, comprising (I) preparing a combination of chitosan and Pt nanoclusters; (II) preparing inclusion bodies of water-soluble antioxidant factors, fat-soluble antioxidant factors and liposomes; (III) adding the inclusion body into the combination body and stirring the mixture thoroughly to obtain a biomimetic high bioactive antioxidant defense liposome system.

5. The method for constructing a biomimetic high bioactive antioxidant defense liposome system according to claim 4, characterized in that: In step (I), the specific steps for preparing the chitosan and Pt nanocluster combination are: adding deionized water to chitosan and adjusting the pH to 7.0 to obtain a chitosan solution; adding deionized water to a soluble Pt salt to obtain a Pt salt solution; The Pt salt solution and the chitosan solution are quickly mixed and stirred to obtain a mixed solution. A reducing agent is added to the mixed solution, the pH is adjusted to 8-12, and stirring is continued for 12-72 hours. The solution after stirring is dialyzed and freeze-dried.

6. The method for constructing a biomimetic high bioactivity antioxidant defense liposome system according to claim 4, characterized in that: In step (II), the water-soluble antioxidant factor is glutathione, and the fat-soluble antioxidant factor is vitamin E. The specific steps of preparing the glutathione, vitamin E and liposome inclusion body are as follows: dissolving egg yolk lecithin, cholesterol and vitamin E, and removing the solvent by rotary evaporation; Add glutathione aqueous solution and hydrate at 37-53°C; The liposomes are extruded using a liposome extruder, and the liposomes are repeatedly passed through a 0.05-0.5 μm filter membrane to obtain inclusions of glutathione and liposomes.

7. The method for constructing a biomimetic high bioactivity antioxidant defense liposome system according to claim 4, characterized in that: In step (I), the raw materials for preparing the combination of chitosan and Pt nanoclusters include chitosan and soluble Pt salt; in step (II), the water-soluble antioxidant factor is glutathione, the fat-soluble antioxidant factor is vitamin E, and the raw materials for preparing the inclusion of glutathione, vitamin E and liposomes include egg yolk lecithin, cholesterol, glutathione and vitamin E; The mass ratio of glutathione to soluble Pt salt is any ratio in the range of 2:1 to 8:1; The mass ratio of egg yolk lecithin to soluble Pt salt is any ratio in the range of 8:1 to 16:1; The mass ratio of egg yolk lecithin to cholesterol is any ratio in the range of 1:1 to 8:1; The mass ratio of egg yolk lecithin to vitamin E is any ratio in the range of 5:1 to 20:1.

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