阅读说明：本技术 一种人工外泌体复合物的制备方法及应用 (Preparation method and application of artificial exosome compound ) 是由 李俊翔 朱才彬 于 2019-03-12 设计创作，主要内容包括：本发明属于生物化学技术领域,具体涉及一种人工外泌体复合物的制备方法及应用。该复合物的制备原料为线性聚乙烯亚胺(PEI)和透明质酸(HA)。本发明的HA-PEI制备过程,可以得到线性的聚乙烯亚胺,而目前大部分聚乙烯亚胺均为支链化的；HA-PEI制备方案,可以定量制备特定分子量的HA-PEI；操作步骤可控,可以规模化工业生产；HA-PEI可通过透明质酸的配体活性及线性聚乙烯亚胺的透膜性,促进皮肤深层细胞对包裹的活性物质的吸收。(The invention belongs to the technical field of biochemistry, and particularly relates to a preparation method and application of an artificial exosome compound. The preparation raw materials of the compound are linear Polyethyleneimine (PEI) and Hyaluronic Acid (HA). In the preparation process of the HA-PEI, linear polyethyleneimine can be obtained, but most of the polyethyleneimine is branched at present; the HA-PEI preparation scheme can quantitatively prepare HA-PEI with a specific molecular weight; the operation steps are controllable, and large-scale industrial production can be realized; the HA-PEI can promote the absorption of the encapsulated active substances by the cells in the deep layer of the skin through the ligand activity of hyaluronic acid and the membrane permeability of linear polyethyleneimine.)

1. An artificial exosome complex, characterized by: the preparation raw materials of the compound are linear Polyethyleneimine (PEI) and Hyaluronic Acid (HA).

2. The artificial exosome complex according to claim 1, characterized in that: the hyaluronic acid is disaccharide unit composed of D-glucuronic acid and N-acetylglucosamine, has molecular weight of 400-1800000, and is in the form of hyaluronic acid or sodium hyaluronate.

3. The artificial exosome complex according to claim 1, characterized in that: the linear polyethyleneimine is obtained by heating and hydrolyzing poly (2-ethyl-2-oxazoline) with sulfuric acid aqueous solution, and the molecular weight is between 1000 and 100000.

4. A method for preparing an artificial exosome complex according to any one of claims 1-3, comprising the steps of:

a.45 g of poly (2-ethyl-2-oxazoline) was dissolved in 200mL of a 30% aqueous solution of sulfuric acid while a heating reflux apparatus was set up.

b. Heating the reaction solution to a reflux state, and detecting the pH value of the distilled solution at the same time, wherein the pH value is mainly used for judging the reaction by-product propionic acid. The propionic acid can be azeotroped with water until no more propionic acid is distilled off and the reaction can be stopped, which takes about 7 days.

c. After the reaction was terminated, the reaction system was cooled to ice bath conditions while being mechanically stirred. And (4) adjusting the pH value to be neutral by using a sodium hydroxide solution, supplementing a part of sodium hydroxide, and excessively adjusting the pH value to be alkaline.

d. During the adjustment of the pH, solids slowly precipitate.

e. Filtered and washed with copious amounts of distilled water. The washing is stopped until the pH of the filtrate is from alkaline to neutral.

f. The solid was filtered off and dried under vacuum at room temperature until no moisture remained, which took approximately 7 days.

g. 600mg of HA is added into 300mL of dd water in a beaker 1 to prepare a solution A, 12g of PEI is added into a beaker 2 to prepare a solution B by adding 300mL of dd water, the solution A is added into the solution B, and 1M HCl is added to adjust the pH value of the solution to be 6.5, so that a solution C is obtained. A mixed solvent of 6.3mmol of EDC and 6.3mmol of HOBt dissolved in 15mL of dd water and 15mL of DMSO was taken to form a solution D, and the solution C and the solution D were added to a 1.5L round-bottomed flask, stirred at room temperature for 24 hours, and adjusted to pH7 with 1M NaOH solution to obtain a reaction solution.

h. The reaction solution was added to an activated dialysis bag (8000- + 14000KD), dialyzed against 100mM NaCl solution for 2 days, dialyzed against 25% by volume of ethanol for 1d, and finally dialyzed against pure water for 1d to obtain HA-PEI solution, which was freeze-dried for two days to obtain white spongy HA-PEI.

i. 10 g of the dried HA-PEI was dispersed in 50mL of distilled water, and a dilute hydrochloric acid solution was added to pH 7.1. Subsequently, distilled water was added to make 100 mL.

And j.0.2 micron filter membrane is used for carrying out aseptic treatment on the solution, so that HA-PEI mother liquor with the concentration of 100mg/mL can be obtained.

5. The artificial exosome complex according to claim 1, characterized in that: also includes amide bonds.

6. The artificial exosome complex according to claim 1, characterized in that: the artificial exosome complex is prepared in the form of aqueous solution, and the concentration is between 1mg/mL and 1 g/mL.

7. Use of an artificial exosome complex, characterized by: the artificial exosome complex is formulated into a formulation that significantly promotes deep skin absorption of active substances through the hyaluronic acid receptors.

8. Use according to claim 7, characterized in that: the preparation is any clinically acceptable preparation.

9. Use according to claim 7, characterized in that: the artificial exosome complex is a preparation of the only active ingredient of the active substance for promoting deep skin absorption, or the complex is used for preparing the preparation of the active substance for promoting skin absorption together with other substances.

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a preparation method and application of an artificial exosome compound.

Background

The effectiveness of skin care products is primarily due to the efficiency of active absorption, and few active delivery vehicles are currently available on the market. The linear polyacetimide has both hydrophilic group and lipophilic group, and can form double molecule layer with hydrophilic group outside the lipophilic group to wrap matter to form microcapsule, which can fuse with cell membrane and deliver inclusion matter into cell. However, the microcapsules are not cell selective because of the membrane fusion characteristics they utilize. Hyaluronic acid, also known as hyaluronic acid, is a natural moisturizing lubricant, and is not effectively absorbed by deep skin cells due to the large molecular weight of hyaluronic acid, so that the skin care durability is limited.

Disclosure of Invention

One technical problem to be solved by one aspect of the present disclosure is to provide a preparation method and an application of a novel artificial exosome complex, aiming at fusing linear polyethyleneimine and hyaluronic acid, and promoting effective absorption of active substances by deep skin by using synergistic effect of ligand activity of hyaluronic acid and membrane-penetrating activity of polyethyleneimine for skin care products.

In order to achieve the purpose, the invention adopts the following technical scheme that the artificial exosome compound is prepared from raw materials of linear Polyethyleneimine (PEI) and Hyaluronic Acid (HA).

Furthermore, the hyaluronic acid is disaccharide unit composed of D-glucuronic acid and N-acetylglucosamine, has a molecular weight of 400-1800000, and is in the form of hyaluronic acid or sodium hyaluronate.

Further, the linear polyethyleneimine is obtained by heating and hydrolyzing poly (2-ethyl-2-oxazoline) through sulfuric acid aqueous solution, and the molecular weight is between 1000 and 100000.

Further, the compound also comprises an amido bond.

Further, the artificial exosome complex is prepared in the form of aqueous solution, and the concentration is 1 mg/mL-1 g/mL.

A preparation method of the artificial exosome compound comprises the following steps:

a.45 g of poly (2-ethyl-2-oxazoline) was dissolved in 200mL of a 30% aqueous solution of sulfuric acid while a heating reflux apparatus was set up.

b. Heating the reaction solution to a reflux state, and detecting the pH value of the distilled solution at the same time, wherein the pH value is mainly used for judging the reaction by-product propionic acid. The propionic acid can be azeotroped with water until no more propionic acid is distilled off and the reaction can be stopped, which takes about 7 days.

c. After the reaction was terminated, the reaction system was cooled to ice bath conditions while being mechanically stirred. And (4) adjusting the pH value to be neutral by using a sodium hydroxide solution, supplementing a part of sodium hydroxide, and excessively adjusting the pH value to be alkaline.

d. During the adjustment of the pH, solids slowly precipitate.

e. Filtered and washed with copious amounts of distilled water. The washing is stopped until the pH of the filtrate is from alkaline to neutral.

f. The solid was filtered off and dried under vacuum at room temperature until no moisture remained, which took approximately 7 days.

g. 600mg of HA is added into 300mL of dd water in a beaker 1 to prepare a solution A, 12g of PEI is added into a beaker 2 to prepare a solution B by adding 300mL of dd water, the solution A is added into the solution B, and 1M HCl is added to adjust the pH value of the solution to be 6.5, so that a solution C is obtained. A mixed solvent of 6.3mmol of EDC and 6.3mmol of HOBt dissolved in 15mL of dd water and 15mL of DMSO was taken to form a solution D, and the solution C and the solution D were added to a 1.5L round-bottomed flask, stirred at room temperature for 24 hours, and adjusted to pH7 with 1M NaOH solution to obtain a reaction solution.

h. Adding the reaction solution into an activated dialysis bag (8000- + 14000KD), dialyzing in 100mM NaCl solution for 2d, dialyzing in 25% ethanol by volume fraction for 1d, finally dialyzing in pure water for 1d to obtain HA-PEI solution, and freeze-drying for two days to obtain white spongy substance HA-PEI.

i. 10 g of the dried HA-PEI was dispersed in 50mL of distilled water, and a dilute hydrochloric acid solution was added to pH 7.1. Subsequently, distilled water was added to make 100 mL.

And j.0.2 micron filter membrane is used for carrying out aseptic treatment on the solution, so that HA-PEI mother liquor with the concentration of 100mg/mL can be obtained.

An application of artificial exosome complex, which is prepared into a preparation capable of remarkably promoting the absorption of active substances into deep skin through a hyaluronic acid receptor.

Furthermore, the preparation is any clinically acceptable preparation.

Further, the artificial exosome complex is a preparation of the only active ingredient of the active substance for promoting deep skin absorption, or the complex is used for preparing the preparation of the active substance for promoting skin absorption together with other substances.

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

1) in the preparation process of the HA-PEI, linear polyethyleneimine can be obtained, and most of the polyethyleneimine is branched at present.

2) The preparation scheme of the HA-PEI can quantitatively prepare the HA-PEI with a specific molecular weight.

3) The method has controllable operation steps and can realize large-scale industrial production.

4) The HA-PEI of the invention can promote the absorption of the encapsulated active substances by the cells in the deep layer of the skin through the ligand activity of hyaluronic acid and the membrane permeability of linear polyethyleneimine.

Drawings

FIG. 1 is a test of skin irritation by artificial exosomes (Control: Control; Exocrinetter: artificial exosome group), according to one aspect of the present disclosure;

FIG. 2 is a graph of the effect of artificial exosomes on Hacat cell morphology (Control: Control; Exocrinetter: artificial exosome group), according to one aspect of the present disclosure;

FIG. 3 is a graph of the proliferative effect of artificial exosomes on Hacat cells (Control: Control; Exocrinetter: artificial exosome group), according to one aspect of the present disclosure;

FIG. 4 is a measurement of the delivery efficiency of the artificial exosomes to pCDNA3.1-eGFP according to one aspect of the present disclosure;

FIG. 5 is an artificial exosome delivery pathway assay result (DAPI: nuclear dye; Exocrinetter: artificial exosome; Mitochondria: Mitochondria) according to one aspect of the present disclosure;

FIG. 6 is a graph of the effect of artificial exosomes on rat skin activity (Control: Control; Exocrinetrator: artificial exosome group) according to one aspect of the present disclosure.

Detailed Description

The invention will be further explained with reference to specific embodiments, without limiting the invention.

In the following examples, unless otherwise specified, the methods used were conventional, and the reagents used were all commercially available.