Water-dispersible polylipoic acid nanosphere and preparation method thereof

Abstract

The invention discloses a water-dispersible polylipoic acid nanosphere and a preparation method thereof, and belongs to the field of polymer nano functional materials. The nanosphere takes polylipoic acid as a unique framework material, and is characterized by being prepared by the following steps: (1) dissolving lipoic acid in an organic solvent to form a uniform solution, and slowly dropwise adding deionized water while stirring at room temperature to induce molecular self-assembly; (2) after dropwise adding, adding a reducing agent, and stirring at 20-50 DEG C for 0.1-2 hours to obtain a polylipoic acid micelle solution; and (3) dialyzing the polylipoic acid micelle solution in deionized water at room temperature for 8-48 hours to obtain the water-dispersible polylipoic acid nanospheres. The obtained polylipoic acid nanospheres are adjustable in particle size, narrow in size distribution and good in water dispersibility. The whole preparation process does not need additional surfactants or stabilizers, conditions are mild, and steps are simple and convenient. The water-dispersible polylipoic acid nanospheres have application prospects in the fields of biomedical nanomaterials, cosmetics, functional coatings and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of high polymer nanofunctional materials, and particularly relates to a water-dispersible nanosphere of lipoic acid and a preparation method thereof, and is used for providing a novel nanocarrier material. BACKGROUND

[0002] Lipoic acid is an endogenous disulfide compound with strong antioxidant activity. Due to its excellent free radical scavenging capacity, metal ion chelating properties, and potential anti-inflammatory and cell signaling pathway regulating biological functions, it has shown broad application prospects in the fields of medicine, health products and functional materials. However, lipoic acid molecules have obvious application limitations: 1) poor water solubility: its inherent hydrophobicity leads to poor dispersion in aqueous environment and low bioavailability; 2) poor stability: it is easily affected by light, heat and oxidation, and especially prone to degradation or uncontrolled polymerization in solution; 3) short in vivo circulation time: free molecules are easily metabolized and cleared. In order to overcome the above defects, constructing lipoic acid into a nanocarrier system is an important strategy in current research. Such nanocarriers can improve solubility, increase stability and prolong circulation time. However, existing technologies mainly focus on encapsulating lipoic acid monomer molecules with carrier materials. Such methods usually have problems such as limited drug loading, potential risks of carrier materials, complex preparation process, high cost and difficulty in scaling up.

[0003] As a bio-based polymer material cross-linked and polymerized from lipoic acid monomers through disulfide bonds, lipoic acid has attracted attention in recent years due to its excellent biocompatibility, degradability, rich surface carboxyl groups, easy functionalization and other characteristics. It itself has the potential to be used as a drug carrier or biomaterial. However, how to efficiently and controllably prepare lipoic acid material with good water dispersibility and nanoscale morphology is a key bottleneck restricting its application. At present, although some documents have reported that lipoic acid can be polymerized under certain conditions, usually large-size precipitates, gel-like agglomerates or irregular block polymers are obtained, which are difficult to stably disperse into nanospheres in aqueous phase. Traditional polymer nanosphere preparation methods usually require the introduction of surfactants, initiators or stabilizers, which may remain and affect the purity and biological safety of the final product, and the steps are complicated. So far, there is no any public document or patent reporting an effective method for directly, mildly and efficiently preparing water-dispersible lipoic acid nanospheres. Therefore, developing a preparation technology with simple process, mild conditions, without surfactants or stabilizers, and directly obtaining water-dispersible lipoic acid nanospheres has an urgent need and great significance for promoting the practical application of lipoic acid material in the fields of biomedicine, cosmetics, functional coatings and the like. The present application is an innovative solution to this technical gap. SUMMARY

[0004] The application aims to provide a water-dispersible nanosphere with lipoic acid as the only skeleton material and a preparation method thereof.

[0005] The core of the technical solution is to include the following steps: (1) 1 part by weight of lipoic acid is dissolved in 10-100 parts by weight of an organic solvent to form a uniform solution; 20-300 parts by weight of deionized water is slowly added to the solution under stirring at room temperature to induce molecular self-assembly; after the addition is completed, 0.05-0.5 parts by weight of a reducing agent is added, and stirring is continued at 20-50°C for 0.1-2 hours to obtain a lipoic acid micelle solution; (2) The lipoic acid micelle solution obtained in step (1) is placed in a dialysis bag and dialyzed in deionized water at room temperature for 8-48 hours to obtain the water-dispersible lipoic acid nanosphere.

[0006] The organic solvent is selected from one of N,N-dimethylformamide, dimethyl sulfoxide, ethanol, methanol, isopropanol, tetrahydrofuran or acetonitrile.

[0007] The reducing agent is selected from one of dithiothreitol, β-mercaptoethanol or tris(2-carboxyethyl) phosphine The molecular weight cut-off of the dialysis bag is 500-3500 Da.

[0008] The chemical reagents are all of chemical purity or above.

[0009] The "room temperature" refers to 20-30°C unless otherwise specified.

[0010] The application innovatively uses lipoic acid as a single component to successfully construct a water-dispersible lipoic acid nanosphere. This innovation not only effectively breaks through the application limitations of traditional lipoic acid and lipoic acid due to hydrophobicity, but also opens up a new path for the preparation of water-dispersible polymer nanospheres, which is expected to inject new vitality into the fields of biomedical nanomaterials, cosmetics and functional coatings, and provide key core material support.

[0011] The technical advantages presented by the application are as follows: 1) The preparation process is simple and efficient, the operating conditions are mild, no additional additives such as surfactants or stabilizers are needed, the production process is greatly simplified, the preparation cost is reduced, and potential problems caused by the addition of other substances are avoided.

[0012] 2) The prepared liposomal ubiquinol nanospheres exhibit excellent water dispersibility and stability. Experiments show that the water dispersion has not yet appeared obvious precipitation phenomenon after standing for a week, which provides a strong guarantee for its long-term stability in practical application.

[0013] 3) Through transmission electron microscope observation and dynamic light scattering instrument detection, the nanospheres can be clearly seen to present spherical structure, and the particle size distribution is relatively concentrated and the size is adjustable. Such morphology and particle size characteristics make it have more extensive adaptability and more excellent performance in subsequent application development.

[0014] Based on the unique performance advantages of the water dispersible liposomal ubiquinol nanospheres, it shows application prospects in the fields of drug carriers, targeted therapy in the biomedical field, functional ingredient transmission, skin care in the cosmetic field, and special performance imparting in the functional coating field. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is a process schematic diagram for preparing water dispersible liposomal ubiquinol nanospheres in example 1 of the present application.

[0016] Figure 2 The figure is a transmission electron microscope photo of water dispersible liposomal ubiquinol nanospheres prepared in example 1 of the present application.

[0017] Figure 3 The figure is a photo of water dispersion liquid of water dispersible liposomal ubiquinol nanospheres prepared in example 1 of the present application.

[0018] Figure 4 The figure is a particle size distribution curve of water dispersible liposomal ubiquinol nanospheres prepared in example 1 of the present application in water.

[0019] Figure 5 The figure is a column chart of cell survival rate of water dispersible liposomal ubiquinol nanospheres (example 1) with different concentrations and B16 cells and L929 cells after co-culturing for 24 hours in a 37℃, 5% CO2 incubator.

[0020] Figure 6 The figure is a transmission electron microscope photo of water dispersible liposomal ubiquinol nanospheres prepared in example 2 of the present application.

[0021] Figure 7 The figure is a particle size distribution curve of water dispersible liposomal ubiquinol nanospheres prepared in example 2 of the present application in water.

[0022] Figure 8 The figure is a transmission electron microscope photo of water dispersible liposomal ubiquinol nanospheres prepared in example 3 of the present application.

[0023] Figure 9The particle size distribution curve of the water-dispersible lipopicolinic acid nanospheres prepared in Example 3 in water. DETAILED DESCRIPTION

[0024] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The intent is to convey the principles and alternative implementations of the present application.

[0025] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each individual intermediate value and each smaller range that falls within the range of values. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described herein.

[0027] Many modifications and variations of this application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0028] The present application discloses a preparation method of water-dispersible lipopicolinic acid nanospheres, comprising the following steps: (1) 1 part by weight of lipopicolinic acid is dissolved in 10-100 parts by weight of organic solvent to form a uniform solution; under room temperature, 20-300 parts by weight of deionized water is slowly added to the solution to induce molecular self-assembly while stirring; after the addition is completed, 0.05-0.5 parts by weight of reducing agent is added, and the stirring is continued at 20-50°C for 0.1-2 hours to obtain a lipopicolinic acid micelle solution; (2) The lipopicolinic acid micelle solution obtained in step (1) is placed in a dialysis bag and dialyzed in deionized water at room temperature for 8-48 hours to obtain water-dispersible lipopicolinic acid nanospheres.

[0029] In some optional embodiments, the organic solvent in step (1) is one of N,N-dimethylformamide, dimethyl sulfoxide, ethanol, methanol, isopropanol, tetrahydrofuran, and acetonitrile.

[0030] In some alternative embodiments, the reducing agent in step (1) is selected from one of dithiothreitol, β-mercaptoethanol, and tris(2-carboxyethyl)phosphine.

[0031] The dialysis bag according to the present application has a molecular weight cut-off of 500-3500 Da.

[0032] The chemical reagent according to the present application is of chemical purity or higher purity.

[0033] In the present application, "room temperature" refers to 20-30°C unless otherwise specified.

[0034] In the following examples, the reagents, materials, and instruments used are commercially available unless otherwise specified.

[0035] Example 1 (1) 40 mg of lipoic acid was dissolved in 2 g of N,N-dimethylformamide to form a uniform lipoic acid solution. Under room temperature, 6 g of deionized water was slowly added to the lipoic acid solution drop by drop to induce molecular self-assembly while stirring. After the addition of deionized water was completed, 10 mg of dithiothreitol was added, followed by continuous stirring at 35°C for 0.5 hours, thereby obtaining a poly-lipoic acid micelle solution.

[0036] (2) The poly-lipoic acid micelle solution obtained in step (1) was placed in a dialysis bag with a molecular weight cut-off of 1800 Da, and dialyzed in deionized water at room temperature for 24 hours, thereby obtaining water-dispersible poly-lipoic acid nanospheres.

[0037] Figure 1 A process diagram for preparing water-dispersible poly-lipoic acid nanospheres according to Example 1 of the present application is shown. First, the disulfide bond of lipoic acid is reduced to a mercapto group by means of a reducing agent, followed by a self-assembly crosslinking reaction in an aqueous phase, thereby realizing the transformation from hydrophobic lipoic acid to hydrophilic poly-lipoic acid nanospheres, and preparing water-dispersible poly-lipoic acid nanospheres.

[0038] Figure 2 A transmission electron microscope photo of the water-dispersible poly-lipoic acid nanospheres prepared according to Example 1 of the present application is shown. It can be clearly observed that the poly-lipoic acid nanospheres exhibit a relatively regular spherical morphology, with a size range of 35-75 nm. Figure 2

[0039] Figure 3 A photo of a water dispersion of the poly-lipoic acid nanospheres prepared according to Example 1 of the present application is shown. It can be seen that the poly-lipoic acid nanospheres can be stably dispersed in water to form a solution with a uniform appearance, exhibiting good water dispersibility. Figure 3

[0040] Figure 4 ​​is the particle size distribution curve of the poly-lipoic acid nanospheres prepared in Example 1 in water, which is determined by a dynamic light scattering instrument. From Figure 4 It can be found that the size distribution of the poly-lipoic acid nanospheres in water is narrow, and the average particle size is 82 nm, further indicating that the nanospheres have a relatively uniform particle size characteristic.

[0041] Figure 5 is the column chart of cell survival rate of B16 cells and L929 cells after being co-cultured with the water-dispersible poly-lipoic acid nanospheres (Example 1) of different concentrations for 24 hours in a 37℃, 5% CO2 incubator. The result is determined by a 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method. From Figure 5 It can be seen from the figure that the cell survival rate of the water-dispersible poly-lipoic acid nanospheres prepared in the application is still 95% at a concentration of 400 μg / ml, indicating that the nanospheres have good biocompatibility.

[0042] Example 2 (1) 40 mg of lipoic acid is dissolved in 2 g of isopropyl alcohol to form a uniform lipoic acid solution. Under room temperature conditions, 4 g of deionized water is slowly added to the lipoic acid solution while stirring to induce molecular self-assembly. After the deionized water is added, 8 mg of dithiothreitol is added, and then stirring is continued at a temperature of 35℃ for 1 hour, thereby preparing a poly-lipoic acid micelle solution.

[0043] (2) The poly-lipoic acid micelle solution obtained in step (1) is placed in a dialysis bag with a molecular weight cut-off of 500 Da, and dialyzed in deionized water at room temperature for 36 hours, thereby preparing water-dispersible poly-lipoic acid nanospheres.

[0044] Figure 6 is a transmission electron microscope photo of the water-dispersible poly-lipoic acid nanospheres prepared in Example 2. From Figure 6 It can be seen that the poly-lipoic acid nanospheres have a regular spherical morphology, and the size range is between 60-170 nm.

[0045] Figure 7 is the particle size distribution curve of the poly-lipoic acid nanospheres prepared in Example 2 in water. The curve is determined by a dynamic light scattering instrument. From Figure 7 It can be seen that the particle size distribution of the poly-lipoic acid nanospheres in water is relatively concentrated, and has a relatively narrow distribution range, and the average particle size is 158 nm.

[0046] Example 3 (1) 50 mg lipoic acid was dissolved in 5 g methanol to form a uniform lipoic acid solution. 15 g deionized water was slowly added dropwise to the lipoic acid solution under stirring at room temperature to induce molecular self-assembly. After the deionized water was added completely, 25 mg β-mercaptoethanol was added, and then the stirring was continued for 0.5 h at 30 °C to obtain a poly-lipoic acid micelle solution.

[0047] (2) The poly-lipoic acid micelle solution obtained in step (1) was placed in a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in deionized water at room temperature for 24 h to obtain water-dispersible poly-lipoic acid nanospheres.

[0048] Figure 8 A transmission electron microscope image of the water-dispersible poly-lipoic acid nanospheres prepared in Example 3 of the present application. It can be seen from the image that the poly-lipoic acid nanospheres have a spherical morphology with a size of 100-200 nm. Figure 8

[0049] Figure 9 A particle size distribution curve of the poly-lipoic acid nanospheres prepared in Example 3 of the present application in water. The curve was determined by a dynamic light scattering instrument. It can be seen from the curve that the size distribution of the poly-lipoic acid nanospheres in water is narrow, and the average particle size is 218 nm. Figure 9

[0050] Example 4 (1) 50 mg lipoic acid was dissolved in 0.5 g dimethyl sulfoxide to form a uniform lipoic acid solution. 1 g deionized water was slowly added dropwise to the lipoic acid solution under stirring at room temperature to induce molecular self-assembly. After the deionized water was added completely, 6 mg β-mercaptoethanol was added, and then the stirring was continued for 2 h at 30 °C to obtain a poly-lipoic acid micelle solution.

[0051] (2) The poly-lipoic acid micelle solution obtained in step (1) was placed in a dialysis bag with a molecular weight cut-off of 1800 Da, and dialyzed in deionized water at room temperature for 36 h to obtain water-dispersible poly-lipoic acid nanospheres.

[0052] Example 5 (1) 50 mg lipoic acid was dissolved in 3 g ethanol to form a uniform lipoic acid solution. 12 g deionized water was slowly added dropwise to the lipoic acid solution under stirring at room temperature to induce molecular self-assembly. After the deionized water was added completely, 20 mg dithiothreitol was added, and then the stirring was continued for 0.1 h at 30 °C to obtain a poly-lipoic acid micelle solution.

[0053] ​​(2) The poly-lipoic acid micelle solution obtained in step (1) was placed in a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in deionized water at room temperature for 12 hours to obtain water-dispersible poly-lipoic acid nanospheres.

[0054] Example 6 (1) 50 mg of lipoic acid was dissolved in 4 g of isopropyl alcohol to form a uniform lipoic acid solution. Under room temperature conditions, 10 g of deionized water was slowly added dropwise to the lipoic acid solution while stirring to induce molecular self-assembly. After the deionized water was added, 2.5 mg of tris(2-carboxyethyl) phosphine was added, and then stirring was continued at a temperature of 50°C for 1.5 hours to obtain a poly-lipoic acid micelle solution.

[0055] (2) The poly-lipoic acid micelle solution obtained in step (1) was placed in a dialysis bag with a molecular weight cut-off of 500 Da, and dialyzed in deionized water at room temperature for 48 hours to obtain water-dispersible poly-lipoic acid nanospheres.

[0056] Example 7 (1) 20 mg of lipoic acid was dissolved in 1 g of tetrahydrofuran to form a uniform lipoic acid solution. Under room temperature conditions, 4 g of deionized water was slowly added dropwise to the lipoic acid solution while stirring to induce molecular self-assembly. After the deionized water was added, 8 mg of β-mercaptoethanol was added, and then stirring was continued at a temperature of 30°C for 1 hour to obtain a poly-lipoic acid micelle solution.

[0057] (2) The poly-lipoic acid micelle solution obtained in step (1) was placed in a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed in deionized water at room temperature for 8 hours to obtain water-dispersible poly-lipoic acid nanospheres.

[0058] Example 8 (1) 100 mg of lipoic acid was dissolved in 6 g of acetonitrile to form a uniform lipoic acid solution. Under room temperature conditions, 15 g of deionized water was slowly added dropwise to the lipoic acid solution while stirring to induce molecular self-assembly. After the deionized water was added, 25 mg of tris(2-carboxyethyl) phosphine was added, and then stirring was continued at a temperature of 20°C for 2 hours to obtain a poly-lipoic acid micelle solution.

[0059] (2) The poly-lipoic acid micelle solution obtained in step (1) was placed in a dialysis bag with a molecular weight cut-off of 1800 Da, and dialyzed in deionized water at room temperature for 15 hours to obtain water-dispersible poly-lipoic acid nanospheres.

[0060] Comparative Example 1 The difference compared with Example 1 is only that the organic solvent is chloroform instead of N,N-dimethylformamide.

[0061] Specifically, 40 mg of lipoic acid was dissolved in 2 g of chloroform, then 6 g of deionized water was added dropwise under the same conditions and 10 mg of dithiothreitol was added, and stirring was continued at 35°C for 0.5 hours. However, it was found during the experiment that the solution was obviously layered and no micelles were formed. After dialysis, no stable dispersed nanospheres were obtained, and the product was precipitated in the form of a flocculent precipitate. This result shows that an organic solvent that is not miscible with water (such as chloroform) cannot effectively induce lipoic acid molecules to self-assemble, and thus cannot prepare water-dispersible lipoic acid nanospheres according to the method of the present application.

[0062] Comparative Example 2 Compared with Example 1, the only difference is that the reducing agent is changed to vitamin C.

[0063] According to the procedure of Example 1, after adding vitamin C, it was found that the reduction of lipoic acid was weak. Part of the lipoic acid could not participate in the formation of lipoic acid nanospheres, and lipoic acid precipitate appeared in the system. Moreover, the structure of the formed lipoic acid nanospheres was not stable enough and was prone to aggregation. This shows that the reducing agent defined in the present application plays an important role in ensuring the formation and stability of the nanospheres.

[0064] Comparative Example 3 Different from Example 1, the feeding ratio of lipoic acid to deionized water was adjusted to 1:1000 (weight ratio, i.e. 40 mg of lipoic acid corresponds to 40 g of deionized water).

[0065] According to the method of Example 1, during the process of inducing molecular self-assembly, due to the excessive dilution of deionized water, the solution concentration was too low to promote the lipoic acid molecules to form stable lipoic acid micelles. The water-dispersible lipoic acid nanospheres obtained after the dialysis step had a wide particle size distribution range, and their stability was not as good as that of the product prepared according to the feeding ratio specified in Example 1, and they were prone to settle in water. It can be seen that precise control of the feeding ratio of lipoic acid to deionized water is one of the key factors to ensure the successful preparation of lipoic acid nanospheres and to ensure their performance.

[0066] Comparative Example 4 Compared with Example 1, the only difference is that the dialysis time is only 2 hours.

[0067] According to the method of Example 1, the self-assembly crosslinking reaction of lipoic acid micelles during dialysis was not sufficient, resulting in a wide particle size distribution of the product and poor stability. In the present application, the self-assembly crosslinking reaction of micelles during dialysis needs to be completed for 8-48 hours. If the dialysis time is too short, the crosslinking is not complete, which will make the particle size distribution of the product wider and the stability lower; while if the dialysis time is too long, it is not necessary. Therefore, selecting the appropriate dialysis time plays an important role in preparing lipoic acid nanospheres with narrow particle size distribution and high stability.

[0068] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art based on the above-described embodiments should fall within the scope of the present application defined by the claims.

Claims

1. A method for preparing water-dispersible nano-spheres of polythioic acid, characterized in that, The method comprises the following steps: (1) dissolving 1 part by weight of lipoic acid in 10-100 parts by weight of an organic solvent to form a uniform solution; slowly adding 20-300 parts by weight of deionized water to the solution to induce molecular self-assembly under room temperature conditions while stirring; after the addition is completed, adding 0.05-0.5 parts by weight of a reducing agent, and continuously stirring at 20-50°C for 0.1-2 hours to obtain a poly-lipoic acid micelle solution; (2) placing the poly-lipoic acid micelle solution obtained in step (1) in a dialysis bag, and dialyzing in deionized water at room temperature for 8-48 hours to obtain water-dispersible poly-lipoic acid nanospheres.

2. The production method according to claim 1, characterized by, The organic solvent is selected from one of N,N-dimethylformamide, dimethyl sulfoxide, ethanol, methanol, isopropanol, tetrahydrofuran or acetonitrile.

3. The preparation method according to claim 1, characterized in that, The reducing agent is selected from one of dithiothreitol, β-mercaptoethanol or tris(2-carboxyethyl)phosphine.

4. The production method according to claim 1, characterized by, The dialysis bag has a molecular weight cut-off of 500-3500 Da.

5. The preparation method according to claim 1, characterized in that, The chemical reagent is of chemical purity or higher purity.

6. Water-dispersible poly-lipoic acid nanospheres prepared by the preparation method of any one of claims 1-5.

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