WO2020037783A1 - THYMOSIN β-4 ETHOSOME AND PREPARATION PROCESS THEREOF - Google Patents

Abstract

Disclosed are a thymosinβ-4 ethosome, a preparation method thereof and uses thereof in preparation of products for wound healing repairing or scar repairing; the thymosinβ-4 ethosome comprises: 0.022％-0.043％ of thymosinβ-4, 0.054％-0.084％ of phospholipid, 0.018％-0.028％ of cholesterol, 0.02％-0.04％ of an anionic surfactant, 10.10％-30.30％ of ethanol, and distilled water as balance.

Description

Thymosin beta-4 alcohol plastid and preparation process thereof Technical field

The invention relates to the field of pharmaceutical preparations, in particular to a thymosin beta-4 alcohol plastid and its preparation process and application.

Background technique

Skin injuries caused by various trauma, burns, surgery, and radiation are common clinical diseases with high incidence. According to statistics, the cost of trauma treatment worldwide reached $ 11.5 billion in 2016. With current treatments, the wound formation rate is as high as 62% (white) and 80% (non-white). These scars often bring long-term physical disorders and psychological burdens to patients. It is of great research significance to develop pharmaceutical preparations that can accelerate wound healing, promote the regeneration of accessory organs such as hair follicles and sweat glands in wounded areas, and help double repair of wound tissues in structure and function. At present, local supplementation of exogenous cytokines to the wound has been shown to play an important role in wound repair of various tissues and organs.

Thymosin β-4 is a cytokine with multiple biological functions. It is a multifunctional peptide consisting of 43 amino acids. It is widely distributed in nucleated cells and is rich in platelets. Its function is similar to that of G-actin muscle. Protein binding is closely related and can regulate actin polymerization and depolymerization. Thymosin β-4 Therefore, thymosin β-4 has potential clinical application value in plastic surgery, and has important functions such as promoting cell differentiation and maturation, tissue wound repair, vascular regeneration, and hair follicle regeneration. It is used in wound repair, corneal repair, and myocardial repair. This area has great potential for drug development, but the application of thymosin β-4 as a scar repair drug has not been well studied.

An alcohol plastid is a lipid bilayer structure composed of phospholipids, cholesterol, ethanol, and water, which is similar to keratinocytes. Its excellent biocompatibility can enclose many drugs that cannot be transdermal. Enters the skin, forms a drug depot, and exerts a local drug effect for a long time. Alcohols have the advantages of higher mobility of the bilayer, easy deformation, better penetration of the skin barrier, and increased drug accumulation in the skin. Compared with liposomes, they have better transdermal absorption performance and higher packaging. Sealing rate, on the one hand, the alcoholic body contains a high concentration of transdermal absorption enhancer ethanol, which changes the tight arrangement of the stratum corneum lipid molecules and enhances lipid fluidity; on the other hand, high concentration of ethanol enhances the alcoholic body membrane The flexibility and fluidity make the encapsulation rate of alcohol-containing liposomes higher. It can deform during the delivery process, overcome the skin's barrier effect, and enhance the penetration ability through the disordered stratum corneum. The application of alcohol plastids to transdermal administration can significantly increase the drug penetration rate and increase the cumulative amount of drugs in the skin. However, there is no related research on the preparation process of thymosin β-4 alcohol plastids in the prior art.

Summary of the Invention

In view of the foregoing prior art, the present invention provides the following technical solutions:

One of the purposes of the present invention is to provide a thymosin β-4 alcohol plastid, which combines thymosin β-4 with an alcohol plastid, and provides a new transdermal preparation, which has good stability and preparation technology. It is simple, has small side effects, and has a stronger transdermal effect. It can enhance the transdermal effect of thymosin β-4 cytokine and enhance the repair effect on scars.

The second purpose of the present invention is to provide the above-mentioned method for preparing thymosin β-4 alcohol plastid. The preparation method in the present invention solves the problem of the stability of the alcohol plastid by adding an anionic surfactant, has low preparation cost and simple process. Good stability.

The third object of the present invention is to protect the preparation of the thymosin β-4 alcohol plastid and use it in scar repair preparations.

In order to achieve the above objective, the present invention adopts the following technical solutions:

The invention provides a thymosin β-4 alcohol substance, which is made of the following raw materials by weight: thymosin β-40.022% to 0.043%, phospholipids 0.054% to 0.084%, cholesterol 0.018% to 0.028%, anionic The surfactant is 0.02% to 0.04%, ethanol is 10.10% to 30.30%, and the rest is distilled water. Anionic surfactants not only play a dispersing role in this solution, they also act as film softeners and have a high degree of deformability. More importantly, the addition of anionic surfactants contributes to the stability of the alcoholic mass. The increase significantly increases the long-term stability of the thymosin β-4 alcohol plastid prepared in the present invention.

Preferably, the raw material composition of the thymosin β-4 alcohol plastid is thymosin β-4 0.025-0.035%, phospholipid 0.06-0.08%, cholesterol 0.02-0.025%, anionic surfactant 0.022-0.03%, and ethanol 18 -28%, the rest is distilled water.

Preferably, the raw material composition of the thymosin β-4 alcohol substance is thymosin β-4 0.03198%, phospholipid 0.06396%, cholesterol 0.02131%, anionic surfactant 0.026665, ethanol 25.23%, and the rest is distilled water.

Further, the aforementioned anionic surfactant is deoxycholate or cholate, and preferably, it is deoxycholate.

In the above raw material composition, the phospholipid may be soybean lecithin or lecithin, and preferably, soybean lecithin.

The present invention also provides a method for preparing the thymosin β-4 alcohol plastid, the steps include:

(1) dissolving phospholipids and cholesterol in absolute ethanol to obtain an alcohol phase solution;

(2) Dissolve the surfactant sodium deoxycholate and thymosin β-4 in distilled water to prepare an aqueous solution;

(3) Slowly inject the aqueous phase solution into the alcohol phase solution for hydration for a certain time under stirring;

(4) The hydration solution is cooled to room temperature, and then passed through a microporous filter to obtain a thymosin β-4 alcohol body.

Preferably, in the step (3), the hydration temperature is 25 to 40 ° C., the hydration time is 10 to 30 min, the stirring rate is 500 to 800 r / min, and the water phase injection rate is 0.2 mL / min to 2 mL / min.

During the research of the present invention, it was found that the injection of the aqueous phase into the alcohol phase is beneficial to obtain a stable and clear solution of the alcoholic mass, and the injection rate and hydration conditions have a significant effect on the encapsulation effect of the alcoholic mass in the present invention.

Preferably, the pore diameter of the microporous filter used in step (4) is 450 nm.

The invention also provides the application of the thymosin β-4 alcohol substance in the preparation of a repairing mask. The repairing mask is composed of the β-4 alcohol body, carbomer gel, and lyophilized protective agent.

The invention also provides a method for preparing the above-mentioned repairing mask. The carbomer gel is placed in distilled water and stirred for a period of time. After the stirring is completed, it is allowed to swell overnight, and then the lyophilized protective agent is slowly added to obtain colorless and transparent. Blank gel matrix. Add the alcohol solution prepared in advance to the blank gel matrix and mix to obtain a β-4 alcohol solution.

Thymosin β-4 alcohol body has a great effect on scar repair and can be used in the field of facial masks to play the dual functions of moisturizing and repairing.

Preferably, the carbomer gel 934 or 940 is placed in distilled water, stirred at 300 to 500 rpm / min for 3 to 5 hours, and left at room temperature overnight after the stirring is completed.

Preferably, the mass ratio of the carbomer gel and the lyophilized protective agent in distilled water is 3 to 5:17 to 20: 0.5 to 2.

Preferably, the lyophilization protection agent is one or a combination of sucrose, glucose and mannitol.

Beneficial effects of the present invention

1. The biological functions of thymosin β-4 include tissue regeneration, remodeling, wound healing, maintaining actin balance, tumor pathogenesis and metastasis, apoptosis and inflammation, etc., and directly use it for scar repair. There are few reports of protective drugs. The present invention chooses to study a thymosin β-4 alcohol plastid and its preparation process, which provides a new research and development idea for scar repair preparations.

2. At present, the plastids mainly contain chemical drugs and traditional Chinese medicines, and rarely contain peptide drugs, mainly due to the large molecular weight of peptide drugs and the low drug loading. However, the topical administration of thymosin β-4 only requires a lower dose to achieve the effect of scar repair. At the same time, the plastids increase the transdermal properties of the drug. In the present invention, thymosin beta-4 is coated with an alcoholic substance, which can reduce the inactivation of the polypeptide and prolong the action time of the active drug.

3. The invention found during the experiment that adding cholesterol during the preparation process is beneficial to the formation of large single-compartment liposomes, and the large hydrophilic cavity is more conducive to encapsulating the hydrophilic drug thymosin β-4. At the same time, cholesterol also exerts With the function of "rivet", the structure of the alcoholic body membrane is stronger. During the research of the present invention, it was also found that the addition of anionic surfactants has a very good improvement effect on the stability of the alcoholic bodies. By adding conventional reagents, unexpected technical effects have been obtained.

4. During the preparation of the alcoholic mass, the amount of reagents, injection method, injection speed, dropping rate, etc. all have important effects on the stability of the alcoholic mass. The present invention provides a method for preparing a stable alcoholic substance through a large amount of research work, and has important promotion significance.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which form a part of this application, are used to provide further understanding of the application. The schematic embodiments of the application and the descriptions thereof are used to explain the application, and do not constitute an improper limitation on the application.

FIG. 1 is a TEM photograph of an alcoholic mass obtained by alcohol phase injection;

FIG. 2 is a TEM photograph of an alcoholic body prepared by water phase injection;

3 is a TEM photograph of an alcoholic body obtained by different preparation methods;

4 is a particle size distribution diagram of β-4 alcohol plastid prepared in Example 4;

5 is a potential distribution diagram of β-4 alcohol plastid prepared in Example 4;

6 is a TEM photograph of a β-4 alcoholic mass prepared in Example 4;

7 is a photographic view of a β-4 alcohol body mask prepared in Example 8;

FIG. 8 is a lyophilized photograph of a β-4 alcohol body mask in Example 8. FIG.

detailed description

The following describes the present invention in detail through examples. It is necessary to point out that the following examples are only for further explanation of the present invention, and should not be construed as limiting the protection scope of the present invention. Those skilled in the art will do according to the content of the following description. Some non-essential improvements and adjustments are within the protection scope of the present invention.

It should be noted that the terminology used herein is only for describing specific embodiments, and is not intended to limit the exemplary embodiments according to the present application. As used herein, the singular forms are intended to include the plural forms as well, unless the context clearly indicates otherwise, and it should also be understood that when the terms "including" and / or "including" are used in this specification, they indicate There are features, steps, operations, devices, components, and / or combinations thereof.

The transmission electron microscope photograph in the present invention was taken by a JEM-2100 transmission electron microscope (JEOL, Japan Electronics Co., Ltd.). The particle size analysis experiment uses a Zetasizer Nano ZS particle size potential analyzer (Malvem UK). The freeze dryer was purchased from Shanghai Yuming Instrument Co., Ltd. (China).

As described in the background art, in view of the shortage of scar repair drugs in the prior art, the present invention proposes a thymosin β-4 alcohol plastid and a preparation method thereof.

In order to enable those skilled in the art to understand the technical solution of the present application more clearly, the technical solution of the present application will be described in detail below with reference to specific embodiments and comparative examples.

Example 1 Selection of preparation method

1.1 Alcohol phase injection

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution, dissolve 0.50 mg of thymosin β-4 in 1.75 mL of distilled water and place in a pear-shaped bottle, and inject the alcohol-phase mixed solution at 400 uL / min into the pear In the flask solution, hydration at 700 r / min for 15 minutes at 30 ° C, and cooling to room temperature through a microporous membrane with a pore diameter of 450 nm, after a certain treatment, the EE was 10 ± 4%, and the appearance of the alcoholic solution was turbid. The background of the TEM photo is dirty and messy, and the shaped alcohol bodies are different in size, and most of them are broken or stuck. The results are shown in FIG. 1.

1.2 Water phase injection

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution and put it in a pear-shaped bottle, dissolve 0.50 mg of thymosin β-4 in 1.75 mL of distilled water, and inject the aqueous mixed solution into the pear-shaped solution at 400 uL / min. In a bottle solution, hydration at 700 r / min for 15 minutes at 30 ° C, after cooling to room temperature, the microporous membrane with a pore diameter of 450 nm was measured. After a certain treatment, the EE was 74 ± 2%. As shown in Figure 2, alcohol content The body solution was clear and transparent with pale blue opalescence and rounded TEM images.

Example 2 Single Factor Test for Preparation of Alcohol

2.1 Cholesterol dosage

Add 1.5mg of soy phospholipid and different amounts of cholesterol to 1mL of absolute ethanol solution and put it in a pear-shaped bottle, dissolve 0.50mg of thymosin β-4 in 1.75mL of distilled water, and inject the aqueous mixed solution into the pear-shaped solution at 400uL / min. In a bottle solution, hydration at 700 r / min for 15 minutes at 30 ° C, and cooling to room temperature through a microporous membrane with a pore diameter of 450 nm, after a certain treatment, EE was measured. The experimental results are shown in the following table:

Effect of cholesterol dosage on EE (n = 3)

The amount of cholesterol has a great effect on the EE of the alcoholic bodies, especially when the cholesterol is not added, the prepared alcoholic body solution cannot show a pale blue opalescence, as shown in Figure 3, TEM photos show Only a very small amount of alcohol is formed and the shape is irregular.

2.2 ethanol concentration

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution and place in a pear-shaped bottle. Dissolve 0.50 mg of thymosin β-4 in 1.75 mL of distilled water. Inject the aqueous phase mixed solution at 400 uL / min. The solution in a pear-shaped bottle was hydrated at 700 rpm for 15 minutes at 30 ° C, and then cooled to room temperature through a microporous membrane with a pore diameter of 450 nm. After a certain treatment, EE was measured. The experimental results are shown in the table below.

Effect of ethanol dosage on EE (n = 3)

The amount of ethanol has a greater impact on the EE of the alcoholic bodies. At the same time, as the amount of ethanol increases, the particle size of the alcoholic bodies also increases. When the ethanol concentration is too high, the TEM photos taken after the alcoholic bodies are placed appear. Many ruptures require further research to explore a reasonable amount of ethanol.

2.3 Beta-4 dosage

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution and place it in a pear-shaped bottle. Dissolve a certain amount of thymosin β-4 in 1.75 mL of distilled water. Inject the aqueous mixed solution into the pear-shaped solution at 400 uL / min. In a bottle solution, hydration at 700 r / min for 15 minutes at 30 ° C, and cooling to room temperature through a microporous membrane with a pore diameter of 450 nm, after a certain treatment, EE was measured. The experimental results are shown in the following table:

Effect of thymosin β-4 dosage on EE (n = 3)

The amount of thymosin β-4 had a great effect on the EE of the protoplasts, and the amount of thymosin β-4 had a greater effect on the stability of the protoplasts.

2.4 Dosage of surfactant

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to different amounts of absolute ethanol solution and put them in pear-shaped bottles. Dissolve 0.50 mg of thymosin β-4 and a certain amount of sodium deoxycholate in 1.75 mL of distilled water. The aqueous mixed solution was injected into the pear-shaped bottle solution at 400uL / min, and hydrated at 700r / min for 15min at 30 ° C. After cooling to room temperature, it passed through a microporous membrane with a pore diameter of 450nm. After a certain treatment, EE was measured. The results are shown in the following table.

Effect of surfactant amount on EE (n = 3)

The EE of alcoholic bodies increases with the increase of the amount of surfactant sodium deoxycholate. Anionic surfactants can penetrate into the skin and interact with intercellular lipids, resulting in increased fluidity of lipid bilayers or lipids. Removal, cytoskeleton structure changes, and the stability of the alkaloids gradually increases, but the zeta potential increases at the same time, and the toxicity and irritation to the skin will increase. Generally, the external transdermal patch has a charge of no more than -65mv, so in the end The dosage of sodium deoxycholate is 6mg, and the zeta potential of the alcoholic body is about -58mv.

2.5 drop rate

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution and put it in a pear-shaped bottle, dissolve 0.50 mg of thymosin β-4 in 1.75 mL of distilled water, and inject the aqueous mixed solution into the pear at different rates. The solution in the flask was hydrated at 700 r / min for 15 minutes at 30 ° C. After cooling to room temperature, the microporous membrane with a pore diameter of 450 nm was measured. After a certain treatment, EE was measured. The experimental results are shown in the following table:

Effect of water injection rate on EE (n = 3)

With the gradual slowing of the water injection rate, the hydration effect is better, and the encapsulation rate of the produced alcoholic mass is greater.

2.6 Hydration speed

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution and put it in a pear-shaped bottle. Dissolve 0.50 mg of thymosin β-4 and a certain amount of sodium deoxycholate in 1.75 mL of distilled water. The phase mixed solution was injected into the pear-shaped bottle solution at 400uL / min, hydrated at a certain speed for 15min at 30 ° C, and cooled to room temperature through a microporous membrane with a pore diameter of 450nm. After a certain treatment, EE was measured. The experimental results As shown in the table below.

Effect of hydration speed on EE (n = 3)

The hydration speed has a great influence on the formation and drug loading of alcohol bodies. Generally speaking, the faster the speed, the better the hydration effect and the better the encapsulation efficiency. Most of the ruptured drugs leaked, which greatly reduced the encapsulation efficiency. TEM photos showed that the alcoholic bodies formed at a stirring rate of 500 to 900 r / min showed less rupture. Almost no alcohol appeared when 700 r / min was selected as the hydration speed. The mass rupture phenomenon, so choose 700r / min as the optimal speed.

2.7 Hydration temperature

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution and put it in a pear-shaped bottle. Dissolve 0.50 mg of thymosin β-4 and a certain amount of sodium deoxycholate in 1.75 mL of distilled water. The phase mixed solution was injected into the pear-shaped bottle solution at 400 uL / min, hydrated at 700 r / min for 15 minutes at a certain temperature, and cooled to room temperature and passed through a microporous membrane with a pore diameter of 450 nm. After a certain treatment, EE was measured. The experimental results are as follows As shown in the table.

Effect of hydration temperature on EE (n = 3)

According to the data in the table, in the range of 25-50 ° C, the hydration temperature does not significantly affect the EE of the alcoholic bodies. Considering that too low and high hydration temperatures are not conducive to the formation of alcoholic bodies, water is used. The melting temperature was 30 ° C.

2.8 Hydration time

Add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution and put it in a pear-shaped bottle. Dissolve 0.50 mg of thymosin β-4 and a certain amount of sodium deoxycholate in 1.75 mL of distilled water. The phase mixed solution was injected into the pear-shaped bottle solution at 400uL / min, and hydrated at 700r / min for 30 minutes at 30 ° C. After cooling to room temperature, it passed through a microporous membrane with a pore diameter of 450nm. After a certain treatment, EE was measured. The results are shown in the following table:

Effect of hydration time on EE (n = 3)

It can be known from the data in the table that the hydration time does not significantly affect the EE of the alcoholic mass. Considering that too long a hydration temperature may damage the stability of the alcoholic mass, the hydration time is determined to be 10 min.

Example 3 Orthogonal Design Method to Optimize Prescriptions

The results of the single-factor experiment in Example 2 indicate that the factors that have a greater effect on alcohol bodies are: the amount of cholesterol (A), the amount of β-4 (B), the concentration of ethanol (C), and the rate of drip (D). The orthogonal design L9 (3 ^ 4) table was used to evaluate each prescription as the parameters of the investigation. The following table is the factor level table and the results of the orthogonal design study.

Orthogonal experimental factors and level table

Orthogonal experiment results

Combine the orthogonal test and single factor experiment to determine the final prescription and process: add 1.5 mg of soy phospholipid and 0.25 mg of cholesterol to 1 mL of absolute ethanol solution, dissolve 0.75 mg of thymosin β-4 in 1.75 mL of distilled water and place in pear In the flask, the alcohol-phase mixed solution was poured into the pear-shaped flask solution at 200 uL / min, and hydrated at 700 r / min for 10 minutes at 30 ° C. After cooling to room temperature, the microporous membrane with a pore diameter of 450 nm was passed.

Example 4 Preparation of β-4 Alcohol

(1) Add 0.02 g of soy phospholipid to 3 mL of absolute ethanol and configure it as phospholipid solution A, add 0.02 g of cholesterol to 1 mL of absolute ethanol and configure it as cholesterol solution B; add 0.01 g of sodium deoxycholate to 10 mL of distilled water Configured as sodium deoxycholate solution C.

(2) Take 0.225mL of solution A, 0.5mL of ethanol and 0.025mL of solution B, mix in a pear-shaped bottle, and seal;

(3) 0.75 mg of thymosin β-4 is dissolved in a mixed solution of 1.125 mL of distilled water and 0.625 mL of solution C;

(4) Inject the solution obtained in step (3) into the pear-shaped bottle solution at 200uL / min, hydrate at 700r / min at 30 ° C for 10min, and cool to room temperature through a microporous membrane with a pore diameter of 450nm to obtain thymosin. β-4 alcohol bodies. As shown in FIG. 4, the obtained thymosin β-4 alcohol plastid has a particle size of 270 ± 3 nm measured by a Malvern dynamic light scattering particle size analyzer; as shown in FIG. 5, the zeta potential is stable at −58 ± 5mv; its microstructure is shown in Fig. 6 transmission electron microscope photograph.

The encapsulation efficiency was determined by centrifugation: the prepared alcohol bodies were placed in an ultracentrifuge at 10,000 r / min for ultracentrifugation for 5 minutes, and 20 uL of the supernatant was used to measure the absorbance at 562 nm using a microplate reader. A standard curve was established to calculate the corresponding concentration. The encapsulation rate calculation formula is EE = (W2-W1) / W2 * 100%, where w1 is the content of free β-4 in the alcohol solution, w2 is the total amount of β-4, and w3 is the total amount of material and β The sum of the total amount of -4, the measured encapsulation rate is 85 ± 5%.

The particle size and PDI of the alcohol bodies prepared by the method of Example 4 were measured, and the stability of the alcohol bodies was measured by the standing method.

Example 5 Preparation of β-4 Alcohol

(1) Add 0.02g of phospholipid to 3mL of absolute ethanol solution, configure as phospholipid solution A, add 0.02g of cholesterol to 1m of absolute ethanol solution, configure as cholesterol solution B; add 0.01g of sodium deoxycholate to 10mL of distilled water Medium is configured as sodium deoxycholate solution C.

(2) Take 0.225mL of solution A, 0.5mL of ethanol and 0.029mL of solution B, mix in a pear-shaped bottle, and seal

(3) Dissolve 0.50 mg of thymosin β-4 in 1.59 mL of distilled water and 0.156 mL of solution C;

(4) Inject the mixed solution obtained in step (3) into the pear-shaped bottle solution at 200uL / min, hydrate at 700r / min at 30 ° C for 10min, and cool to room temperature through a microporous membrane with a pore diameter of 450nm to obtain the thymus. Β-4 alcohol plastid.

The particle size and PDI of the alcoholic bodies prepared by the method of Example 5 were measured, and the stability of the alcoholic bodies was measured by the standing method.

Example 6 Preparation of β-4 Alcohol

(1) 0.02 g of phospholipid was added to 3 mL of absolute ethanol to prepare phospholipid solution A, and 0.02 g of cholesterol was added to 1 mL of absolute ethanol to prepare cholesterol solution B.

(2) Take 0.225mL of solution A, 0.5mL of ethanol and 0.025mL of solution B, mix in a pear-shaped bottle, and seal

(3) 0.75 mg of thymosin β-4 is dissolved in 1.75 mL of distilled water;

(4) Inject the mixed solution obtained in step (3) into the pear-shaped bottle solution at 200uL / min, hydrate at 700r / min at 30 ° C for 10min, and cool to room temperature through a microporous membrane with a pore diameter of 450nm to obtain the thymus. Β-4 alcohol plastid.

The particle size and PDI of the alcoholic bodies prepared by the method of Example 6 were measured, and the stability of the alcoholic bodies was measured by the standing method. After measurement and observation, the comparison of the results of Examples 4, 5 and 6 is shown in the following table:

Example 7 Investigation of Physicochemical Properties of Alcohol

7.1 Pattern

Take an appropriate amount of PRO liposomes in a vial, and observe its appearance to show a pale blue opalescence. Then take one or two drops of alcoholic body solution, dilute appropriately with distilled water, add dropwise to the copper sheet, stain with 2% phosphotungstic acid solution, dry for 30min, and observe the microscopic morphology by transmission electron microscope. As shown in Figure 6, the morphology of the alcoholic substance is round, the surface is smooth and non-adherent, and the particle size distribution is uniform. Obvious fingerprint-like patterns can be observed, all of which are large hydrophilic chambers suitable for hydrophilic Large single-compartment alcohol bodies of sex drugs.

7.2 Particle size and potential

Take an appropriate amount of the prepared liposome solution, dilute it by an appropriate multiple and use a dynamic light scattering instrument to measure the particle size using a Malvern laser particle size analyzer. The particle size is 270 ± 3nm; the zeta potential is stable at -58 ± 5mv.

7.3 Investigation of Stability of Alcohol

Samples 1 and 2 were prepared according to the method of Example 4, and the particle sizes and PDI at 1d, 10d, and 20d were examined, as shown in the following table:

Analysis: Alcohol bodies were left for 10 days and 20 days. Neither particle size nor PDI changed significantly, and the stability was good.

7.4 Investigation of the transdermal properties of β-4 alcohol bodies

Take the skin of the rat's back and sandwich it between the diffuser cover (donor) and the diffuser (receiver). The inner surface of the skin is immersed in isotonic solution of physiological saline, and 2mL of β-4 alcohol solution and free β are taken. -4 drug was immersed in 5 mL of physiological saline for transdermal diffusion. At 1h, 2h, 4h, 6h, 8h, 10h, and 12h, 0.4mL of the solution was measured from the absorption tank, and the same amount of physiological saline was added. The cumulative permeation amount (Q) per unit area of the two was measured with a microplate reader.

Experimental results:

Analysis: Compared to β-4 alcohol plastids and free drugs of equal concentration, the cumulative permeation per unit area is larger and the transdermal performance is better.

Example 8 Alcohol Body Mask

As a very important external dosage form, the gel is widely used in skin administration, which can increase the retention of the drug on the skin surface. The gel contains water-based and oil-based gelling agents, and the alcoholic body is an aqueous liquid. Therefore, in the present invention, gel-carbomer with an aqueous property is selected as the gel matrix of the liposome. This gel has the advantages of good spreadability, no oiliness, good adhesion to the skin, no irritation, easy cleaning, etc. It is widely used in gelling agents for drugs and cosmetics.

8.1 Preparation method

(1) Preparation of alcoholic bodies: preparing alcoholic bodies according to the method described above;

(2) Take 0.5g of carbomer gels 934 and 940, put them in 24.5g of distilled water, stir magnetically at 400rpm / min for 3.5h, and stir at room temperature overnight, fully swell, then slowly add 0.125g lyophilization The protective agent (sucrose, glucose, mannitol, etc.) can be stirred to obtain a colorless and transparent blank gel matrix. Take the alcohol solution prepared in advance and slowly add it to the blank gel matrix, grind and homogenize to obtain β-4 alcohol mass gel.

The β-4 alcohol substance in Example 4 was added to 0.5 g of carbomer gels 934 and 940, placed in 24.5 g of distilled water, and magnetically stirred at 400 rpm / min for 3.5 h. After stirring, it was left at room temperature overnight, and fully swelled. Then, slowly add 0.125g of lyophilized protective agent (sucrose, glucose, mannitol, etc.) and stir to obtain a colorless and transparent blank gel matrix. Take the alcohol solution prepared in advance and slowly add it to the blank gel matrix, grind and homogenize to obtain β-4 alcohol mass gel.

As shown in FIG. 7, the obtained β-4 alcoholic body gel has a uniform color, a translucent shape, and a neutral pH. It was applied to the skin of the back of rabbits for 4 hours, and observed with naked eyes at 30min, 1h, 24h, and 72h after drug removal. The skin on the back of rabbits did not cause adverse reactions such as redness and swelling.

8.2 Appearance

The obtained β-4 alcoholic body gel has a uniform and uniform color, is translucent, and has a neutral pH. It was applied to the skin of the back of rabbits for 4 hours, and observed with naked eyes at 30min, 1h, 24h, and 72h after drug removal. The skin on the back of rabbits did not cause adverse reactions such as redness and swelling.

Although the specific embodiments of the present invention are described above with reference to the accompanying drawings, they are not a limitation on the protection scope of the present invention. Those skilled in the art should understand that based on the technical solution of the present invention, those skilled in the art do not need to make creative work. Various modifications or variations that can be made are still within the protection scope of the present invention.

Claims (10)

1. A thymosin β-4 alcohol plastid is characterized in that it is made of the following raw materials by weight: thymosin β-4 0.022% to 0.043%, phospholipids 0.054% to 0.084%, cholesterol 0.018% to 0.028%, Anionic surfactants are 0.02% to 0.04%, ethanol is 10.10% to 30.30%, and the rest is distilled water.
2. The alcoholic substance according to claim 1, wherein the mass fraction ratio of the raw materials is: thymosin β-4 0.03198%, phospholipid 0.06396%, cholesterol 0.02131%, anionic surfactant 0.02665%, ethanol 25.23%, the rest is distilled water.
3. The thymosin β-4 alcohol plastid according to claim 1, wherein the anionic surfactant is sodium deoxycholate or sodium cholate.
4. The alcoholic body according to claim 1, wherein the phospholipid is soy phospholipid or lecithin, and preferably is soy phospholipid.
5. The method for preparing thymosin β-4 alcohol plastid according to claim 1, comprising the steps of:

   (1) dissolving phospholipids and cholesterol in absolute ethanol to obtain an alcoholic phase solution, the phospholipids are soy phospholipids or lecithin, preferably, soy phospholipids;

   (2) dissolving an anionic surfactant and thymosin β-4 in distilled water to prepare an aqueous phase solution, the surfactant is an anionic surfactant; preferably, sodium deoxycholate or sodium cholate;

   (3) Slowly inject the aqueous phase solution into the alcohol phase solution for hydration under stirring;

   (4) The hydrated solution is cooled to room temperature and passed through a microporous filter to obtain a thymosin β-4 alcohol substance, the pore diameter of the microporous filter is 450 nm.
6. The preparation method according to claim 5, wherein the concentration of the ethanol solution in the mixed solution obtained in step (3) is 25% to 40%.
7. The preparation method according to claim 5, wherein the stirring rate in step (3) is 500-800 r / min
8. The method according to claim 5, wherein the injection rate of the aqueous phase in step (3) is 0.2 mL / min to 2 mL / min.
9. The preparation method according to claim 5, wherein the hydration time in step (3) is 10-30 minutes, and the hydration temperature is 25-40 ° C.
10. Use of the thymosin β-4 alcohol plastid according to claims 1-4 in the field of preparing repairing mask.

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