CN108721604B - Thymosin beta-4 ethosome and preparation process thereof - Google Patents
Thymosin beta-4 ethosome and preparation process thereof Download PDFInfo
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- CN108721604B CN108721604B CN201810951630.5A CN201810951630A CN108721604B CN 108721604 B CN108721604 B CN 108721604B CN 201810951630 A CN201810951630 A CN 201810951630A CN 108721604 B CN108721604 B CN 108721604B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2292—Thymosin; Related peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
Abstract
The invention discloses a thymosin beta-4 ethosome and a preparation method thereof, wherein the thymosin beta-4 ethosome comprises the following specific components: thymosin beta-40.022-0.043%, phospholipid 0.054-0.084%, cholesterol 0.018-0.028%, anionic surfactant 0.02-0.04%, ethanol 10.10-30.30%, and the balance of distilled water; has the technical effects of increasing the transdermal absorption of medicaments and enhancing the repair of the thymosin beta-4 to scar skin. The preparation method of the thymosin beta-4 ethosome solves the problem of stability of the thymosin beta-4 ethosome by adding the anionic surfactant and the cholesterol, and the thymosin beta-4 ethosome obtained by the preparation method has high encapsulation rate, good stability, simple process and good economic benefit. Correspondingly, the invention also protects the application of the thymosin beta-4 ethosome in the field of scar repair.
Description
Technical Field
The invention relates to the field of pharmaceutical preparations, in particular to a thymosin beta-4 ethosome, a preparation process and application thereof.
Background
Skin injuries caused by various wounds, burns, operations, radiation and the like are common clinical diseases and are high in morbidity. Statistically, the cost of wound treatment worldwide reaches $ 115 million in 2016. With the current treatment means, the formation rate of the healed wound surface is as high as 62 percent (caucasian) and 80 percent (non-caucasian), and the scars often bring long-term physiological disorder and psychological burden to patients. The research on the pharmaceutical preparation which can accelerate wound healing, promote the regeneration of accessory organs such as local hair follicles and sweat glands of the wound and help wound tissues to obtain double repair on structure and function has great research significance. Currently, local supplementation of exogenous cytokines to wounds has been shown to play an important role in wound repair in various tissue organs.
Thymosin beta-4 is a cytokine with multiple biological functions, is a multifunctional polypeptide consisting of 43 amino acids, is widely distributed in nucleated cells and enriched in platelets, has functions closely related to the binding of G-actin, and can regulate actin polymerization and depolymerization. Therefore, the thymosin beta-4 has potential clinical application value for plastic surgery, has important functions of promoting cell differentiation maturation, tissue wound repair, blood vessel regeneration, hair follicle regeneration and the like, has great drug development potential in the aspects of wound repair, cornea repair and myocardial repair, but few people perform related research on the application of the thymosin beta-4 directly as a scar repair drug.
The ethosome is composed of phospholipid, cholesterol, ethanol and water, has a lipid bilayer structure similar to stratum corneum cells, can wrap a plurality of medicines which cannot be permeated through the skin by self through excellent biocompatibility, and is carried into the skin to form a medicine storage reservoir to play a local medicine effect persistently. The ethosome has the advantages of higher bilayer fluidity, easy deformation, contribution to penetrating a skin barrier and increasing the accumulation of the drug in the skin, and has better transdermal absorption performance and higher entrapment rate compared with the liposome, on one hand, the ethosome contains a high-concentration transdermal absorption enhancer ethanol, so that the close arrangement of stratum corneum lipid molecules is changed, and the lipid fluidity is enhanced; on the other hand, the high-concentration ethanol enhances the flexibility and the fluidity of the alcoholic membrane, so that the drug encapsulation rate of the alcoholic liposome is higher, the drug can deform in the transfer process, the barrier effect of the skin is overcome, the disordered stratum corneum is penetrated, and the permeability is enhanced. The ethosome is applied to transdermal administration, so that the drug permeation rate can be obviously improved, the accumulation amount of the drugs in the skin is increased, and related researches on a thymosin beta-4 ethosome preparation process are not available in the prior art.
Disclosure of Invention
Aiming at the prior art, the invention provides the following technical scheme:
one of the purposes of the invention is to provide a thymosin beta-4 ethosome, wherein thymosin beta-4 is combined with the ethosome, and a novel transdermal administration preparation is provided, and the preparation has the characteristics of good stability, simple preparation process, small side effect and stronger transdermal effect, can enhance the transdermal effect of thymosin beta-4 cytokines, and improves the repair effect on scars.
The second purpose of the invention is to provide the preparation method of the thymosin beta-4 ethosome, the preparation method solves the stability problem of the ethosome by adding the anionic surfactant, and the preparation method has low preparation cost, simple process and good stability.
The invention also aims to protect the application of the thymosin beta-4 ethosome preparation in the aspect of scar repairing preparations.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a thymosin beta-4 ethosome which is prepared from the following raw materials in parts by weight: thymosin beta-40.022-0.043%, phospholipid 0.054-0.084%, cholesterol 0.018-0.028%, anionic surfactant 0.02-0.04%, ethanol 10.10-30.30% and distilled water in balance. The anionic surfactant can play a role in dispersion and serve as a membrane softener to enable the membrane softener to have high deformability, and more importantly, the addition of the anionic surfactant is beneficial to the improvement of the stability of the ethosome, so that the long-term stability of the thymosin beta-4 ethosome prepared by the invention is obviously improved.
Preferably, the thymosin beta-4 ethosome comprises the raw materials of thymosin beta-40.025-0.035%, phospholipid 0.06-0.08%, cholesterol 0.02-0.025%, anionic surfactant 0.022-0.03%, ethanol 18-28%, and the balance of distilled water.
Preferably, the thymosin beta-4 ethosome comprises thymosin beta-40.03198, phospholipid 0.06396, cholesterol 0.02131, anionic surfactant 0.02665, ethanol 25.23 and the balance of distilled water.
Further, the anionic surfactant is deoxycholate or cholate, and preferably deoxycholate.
The phospholipid in the raw material composition can be soybean phospholipid or lecithin, and is preferably soybean phospholipid.
The invention also provides a preparation method of the thymosin beta-4 ethosome, which comprises the following steps:
(1) dissolving phospholipid and cholesterol in absolute ethyl alcohol to obtain an alcohol phase solution;
(2) dissolving surfactant sodium deoxycholate and thymosin beta-4 in distilled water to prepare aqueous solution;
(3) slowly injecting the water phase solution into the alcohol phase solution under stirring for hydration for a certain time;
(4) cooling the hydrated solution to room temperature, and then filtering the hydrated solution through a microporous filter membrane to obtain the thymosin beta-4 ethosome.
Preferably, the hydration temperature in the step (3) is 25-40 ℃, the hydration time is 10-30 min, the stirring speed is 500-800 r/min, and the injection speed of the water phase is 0.2-2 mL/min.
In the research process of the invention, the injection of the water phase into the alcohol phase is beneficial to obtaining a stable and clear ethosome solution, and the injection rate, the hydration conditions and the like have obvious influence on the encapsulation effect of the ethosome.
Preferably, the pore size of the microporous filter membrane used in step (4) is 450 nm.
The invention also provides application of the thymosin beta-4 ethosome in preparation of a repair mask. The repair mask consists of the beta-4 ethosome, carbomer gel and a freeze-drying protective agent.
The invention also provides a preparation method of the repairing facial mask, which comprises the steps of putting carbomer gel into distilled water, stirring for a period of time, standing overnight after stirring is finished, fully swelling the carbomer gel, and slowly adding the freeze-drying protective agent to obtain the colorless and transparent blank gel matrix. And adding the prepared ethosome solution into the blank gel matrix, and uniformly mixing to obtain the beta-4 ethosome repairing mask.
The thymosin beta-4 ethosome has a great effect on scar repair, can be applied to the field of facial masks, and has double effects of moisturizing and repairing.
Preferably, the carbomer gel 934 or 940 is placed in distilled water, stirred at 300-500 rpm/min for 3-5 hours, and placed at room temperature overnight after stirring.
Preferably, the mass ratio of the carbomer gel to the distilled water freeze-drying protective agent is 3-5: 17-20: 0.5-2.
Preferably, the lyoprotectant is one or a combination of sucrose, glucose and mannitol.
The invention has the advantages of
1. The biological functions of thymosin beta-4 include tissue regeneration, remodeling, wound healing, actin balance maintenance, tumor onset and metastasis, apoptosis action and inflammation, etc., and the application of thymosin beta-4 as scar repairing medicine is reported.
2. At present, the ethosome mainly contains more chemical drugs and traditional Chinese medicine components, and rarely contains polypeptide drugs, mainly because the polypeptide drugs have large molecular weight and the drug-loading rate is too low. However, the thymosin beta-4 local administration can achieve the effect of scar repair only by using a lower dose, meanwhile, the ethosome increases the transdermal property of the medicament, so that the dosage form and the medicament complement each other, and the polypeptide is easy to inactivate.
3. In the experimental process, the invention discovers that the addition of cholesterol in the preparation process is favorable for forming a large single-chamber liposome, the hydrophilic cavity is larger and is more favorable for wrapping the hydrophilic medicament thymosin beta-4, and simultaneously, the cholesterol also plays a role of a rivet so as to enable the structure of an alcohol mass membrane to be firmer. In the research process, the invention also finds that the addition of the anionic surfactant has a good improvement effect on the stability of the ethosome, and the unexpected technical effect is achieved by the addition of the conventional reagent.
4. In the preparation process of the ethosome, the dosage, injection mode, injection speed, dropping rate and the like of the reagent have important influence on the stability of the ethosome. Through a large amount of research work, the invention provides a method for preparing an ethosome with good stability, and the method has important popularization significance.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a TEM photograph of an alcohol phase injected into a prepared ethosome;
FIG. 2 is a TEM photograph of an ethosome prepared by aqueous phase injection;
FIG. 3 is a TEM photograph of ethosome prepared by different preparation methods;
FIG. 4 is a plot of the particle size distribution of the β -4 ethosomes prepared in example 4;
FIG. 5 is a graph of the betaplast potential profile of the alcoholates prepared in example 4;
FIG. 6 is a TEM photograph of the β -4 ethosome prepared in example 4;
FIG. 7 is a photograph of a beta-4 ethosome facial mask prepared in example 8;
FIG. 8 is a photograph of a lyophilized mask of the beta-4 ethosome according to example 8.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that some insubstantial modifications and adaptations by those skilled in the art based on the following descriptions are within the scope of the present invention.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The transmission electron micrograph of the present invention was taken with a JEM-2100 transmission electron microscope (JEOL, Japan Electron Ltd.). Particle size analysis experiments a Zetasizer Nano ZS particle size potential analyzer (malmem britain). The freeze dryer was purchased from shanghai yuming instruments ltd (china).
As introduced in the background art, the invention provides a thymosin beta-4 ethosome and a preparation method thereof aiming at the defects of scar repairing medicaments in the prior art.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific examples and comparative examples.
EXAMPLE 1 selection of preparation methods
1.1 alcohol phase injection
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, dissolving 0.50mg of thymosin beta-4 in 1.75mL of distilled water, placing the solution in a pear-shaped bottle, injecting an alcohol phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at 30 ℃ at 700r/min, cooling to room temperature, passing through a microporous filter membrane with the aperture of 450nm, measuring the EE to be 10 +/-4% after certain treatment, leading the appearance of an ethosome solution to be turbid, leading the background of TEM photographs to be dirty, leading molded ethosomes to be different in size and most of the formed ethosomes to be broken or adhered, and leading the result to be shown in figure 1. 1.2 aqueous injection
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, placing the solution in a pear-shaped bottle, dissolving 0.50mg of thymosin beta-4 in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at 30 ℃ at 700r/min, cooling to room temperature, passing through a microporous filter membrane with the aperture of 450nm, and measuring EE to be 74 +/-2% after certain treatment, wherein the ethosome solution has clear and transparent appearance, light blue opalescence and round TEM picture as shown in figure 2.
Example 2 preparation of ethosome Single factor assay
2.1 amount of Cholesterol
Adding 1.5mg of soybean phospholipid and cholesterol with different dosages into 1mL of absolute ethanol solution, placing the solution into a pear-shaped bottle, dissolving 0.50mg of thymosin beta-4 in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, passing through a microfiltration membrane with the aperture of 450nm, measuring EE after certain treatment, wherein the experimental measurement results are shown in the following table:
effect of Cholesterol dosage on EE (n-3)
The amount of cholesterol had a great influence on EE of the ethosome, and particularly, the obtained ethosome solution did not exhibit a bluish opalescence well without the addition of cholesterol, as shown in fig. 3, the TEM photograph showed that only a very small amount of ethosomes were molded and had irregular shapes.
2.2 ethanol concentration
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, placing the solution in a pear-shaped bottle, dissolving 0.50mg of thymosin beta-4 in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, passing through a microporous filter membrane with the aperture of 450nm, measuring EE after certain treatment, and the experimental measurement results are shown in the following table.
Effect of ethanol dosage on EE (n ═ 3)
The ethanol dosage has a great influence on EE of the ethosome, the particle size of the ethosome is increased along with the increase of the ethanol dosage, and a TEM picture taken after the ethosome is placed has a lot of cracks when the ethanol concentration is too high, so that subsequent research is needed for discussing a reasonable ethanol dosage.
2.3 beta-4 in an amount
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, placing the solution in a pear-shaped bottle, dissolving a certain amount of thymosin beta-4 in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, passing through a microfiltration membrane with the aperture of 450nm, carrying out certain treatment, and measuring EE, wherein the experimental measurement results are shown in the following table:
effect of thymosin beta-4 dosage on EE (n-3)
The amount of thymosin beta-4 has a great influence on the EE of the ethosome, and the amount of thymosin beta-4 has a great influence on the stability of the ethosome.
2.4 amount of surfactant
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into anhydrous ethanol solutions with different amounts, placing the anhydrous ethanol solutions in pear-shaped bottles, dissolving 0.50mg of thymosin beta-4 and a certain amount of sodium deoxycholate in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at 30 ℃ at 700r/min, cooling to room temperature, passing through a microfiltration membrane with the aperture of 450nm, carrying out certain treatment, and measuring EE, wherein the experimental measurement results are shown in the following table.
Effect of surfactant amount on EE (n-3)
The EE of the ethosome is increased along with the increase of the dosage of the surfactant sodium deoxycholate, the anionic surfactant can permeate into the skin and react with intercellular lipid, so that the mobility of a lipid bilayer is increased or lipid is removed, the framework structure of cell protein is changed, the stability of the ethosome in placement is gradually enhanced, but the zeta potential is simultaneously enhanced, the toxicity and the irritation to the skin are increased, the electrification of an external transdermal patch is not more than-65 mv generally, so that the dosage of the final sodium deoxycholate is 6mg, and the zeta potential of the ethosome is about-58 mv at the moment.
2.5 dropping Rate
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, placing the solution in a pear-shaped bottle, dissolving 0.50mg of thymosin beta-4 in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at different rates, hydrating for 15min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, passing through a microfiltration membrane with the aperture of 450nm, carrying out certain treatment, and measuring EE, wherein the experimental measurement results are shown in the following table:
effect of Water injection Rate on EE (n ═ 3)
Along with the gradual slowing of the water injection rate, the hydration effect is better, and the encapsulation rate of the prepared ethosome is higher.
2.6 hydration speed
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, placing the solution in a pear-shaped bottle, dissolving 0.50mg of thymosin beta-4 and a certain amount of sodium deoxycholate in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at a certain rotating speed at 30 ℃, cooling to room temperature, passing through a microfiltration membrane with the aperture of 450nm, carrying out certain treatment, and measuring EE, wherein the experimental measurement results are shown in the following table.
Effect of hydration speed on EE (n-3)
The hydration rotation speed has great influence on the formation and drug loading of the ethosome, generally, the faster the rotation speed, the better the hydration effect, the better the encapsulation efficiency, but the too fast rotation speed can cause most of the rupture drugs of the prepared ethosome to leak, thereby greatly reducing the encapsulation efficiency, TEM photographs show that the formed ethosome has less rupture when the stirring speed is 500-900 r/min, and the ethosome has no rupture phenomenon when the hydration rotation speed is 700r/min, so the 700r/min is selected as the optimal rotation speed.
2.7 hydration temperature
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, placing the solution in a pear-shaped bottle, dissolving 0.50mg of thymosin beta-4 and a certain amount of sodium deoxycholate in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for 15min at a certain temperature of 700r/min, cooling to room temperature, passing through a microfiltration membrane with the aperture of 450nm, carrying out certain treatment, and measuring EE, wherein the experimental measurement results are shown in the following table.
Effect of hydration temperature on EE (n-3)
As can be seen from the data in the table, the hydration temperature does not significantly affect the EE of the ethosome within the range of 25-50 ℃, and the hydration temperature is 30 ℃ considering that the forming of the ethosome is not facilitated by the excessively low hydration temperature and the excessively high hydration temperature. 2.8 hydration time
Adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, placing the solution in a pear-shaped bottle, dissolving 0.50mg of thymosin beta-4 and a certain amount of sodium deoxycholate in 1.75mL of distilled water, injecting the water-phase mixed solution into the pear-shaped bottle solution at 400uL/min, hydrating for a certain time at 30 ℃ at 700r/min, cooling to room temperature, passing through a microfiltration membrane with the aperture of 450nm, carrying out certain treatment, and measuring EE, wherein the experimental measurement results are shown in the following table:
effect of hydration time on EE (n-3)
As can be seen from the data in the table, the hydration time does not significantly affect the EE of the ethosome, and considering that too long a hydration temperature may destroy the stability of the ethosome, the hydration time is determined to be 10 min.
Example 3 orthogonal design optimization recipe
The results of the single-factor experiment in example 2 show that the factors having large influence on ethosome are as follows: the dosage of cholesterol (A), the dosage of beta-4 (B), the ethanol concentration (C) and the dropping rate (D) are evaluated as the examined parameters through an orthogonal design L9(3^4) table, and the following table is a factor level table and the examined result of the orthogonal design.
Orthogonal experiment factor and level meter
Results of orthogonal experiments
The final prescription and process are determined by combining the orthogonal test and the single-factor test as follows: adding 1.5mg of soybean phospholipid and 0.25mg of cholesterol into 1mL of absolute ethanol solution, dissolving 0.75mg of thymosin beta-4 in 1.75mL of distilled water, placing the solution in a pear-shaped bottle, injecting the alcohol-phase mixed solution into the pear-shaped bottle solution at 200uL/min, hydrating for 10min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, and passing through a microfiltration membrane with the aperture of 450 nm.
EXAMPLE 4 preparation of beta-4 Alosomes
(1) Adding 0.02g of soybean phospholipid into 3mL of absolute ethyl alcohol to prepare a phospholipid solution A, and adding 0.02g of cholesterol into 1mL of absolute ethyl alcohol to prepare a cholesterol solution B; 0.01g of sodium deoxycholate was added to 10mL of distilled water to prepare a sodium deoxycholate solution C.
(2) Uniformly mixing 0.225mL of the solution A, 0.5mL of ethanol and 0.025mL of the solution B in a pear-shaped bottle, and sealing;
(3) dissolving 0.75mg of thymosin beta-4 in a mixed solution of 1.125mL of distilled water and 0.625mL of solution C;
(4) and (3) injecting the solution obtained in the step (3) into a pear-shaped bottle solution at 200uL/min, hydrating for 10min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, and then passing through a microfiltration membrane with the aperture of 450nm to obtain the thymosin beta-4 ethosome. As shown in figure 4, the prepared thymosin beta-4 ethosome has a particle size of 270 +/-3 nm measured by a Malvern dynamic light scattering particle sizer; as shown in FIG. 5, the zeta potential is stabilized at-58. + -. 5 mv; the microstructure is shown in the transmission electron micrograph of figure 6.
Encapsulation efficiency was determined by centrifugation: the prepared ethosome is placed in an ultracentrifuge for 5min at 10000r/min, 20uL of supernatant is absorbed and absorbance is measured by using an enzyme-labeling instrument at 562nm, and a standard curve is established to calculate the corresponding concentration. The encapsulation efficiency was calculated as EE ═ 100% W2-W1)/W2, where W1 is the free β -4 content in the ethosome solution, W2 is the total amount of β -4, W3 is the sum of the total amount of material and the total amount of β -4, and the encapsulation efficiency was found to be 85 ± 5%.
The particle size and PDI of the ethosome prepared in example 4 were measured, and the stability of the ethosome was measured by the standing method.
EXAMPLE 5 preparation of beta-4 Alosomes
(1) Adding 0.02g of phospholipid into 3mL of absolute ethyl alcohol solution to prepare a phospholipid solution A, and adding 0.02g of cholesterol into 1m of absolute ethyl alcohol solution to prepare a cholesterol solution B; 0.01g of sodium deoxycholate was added to 10mL of distilled water to prepare a sodium deoxycholate solution C.
(2) Mixing 0.225mL solution A, 0.5mL ethanol and 0.029mL solution B, sealing
(3) Dissolving 0.50mg of thymosin beta 4 in 1.59mL of distilled water and 0.156mL of solution C;
(4) and (4) injecting the mixed solution obtained in the step (3) into a pear-shaped bottle solution at 200uL/min, hydrating for 10min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, and then passing through a microfiltration membrane with the aperture of 450nm to obtain the thymosin beta-4 ethosome.
The particle size and PDI of the ethosome prepared in example 5 were measured, and the stability of the ethosome was measured by the standing method.
EXAMPLE 6 preparation of beta-4 Alosomes
(1) Phospholipid solution A was prepared by adding 0.02g of phospholipid to 3mL of absolute ethanol, and cholesterol solution B was prepared by adding 0.02g of cholesterol to 1mL of absolute ethanol.
(2) Mixing 0.225mL solution A, 0.5mL ethanol and 0.025mL solution B, sealing
(3) Dissolving 0.75mg of thymosin beta-4 in 1.75mL of distilled water;
(4) and (4) injecting the mixed solution obtained in the step (3) into a pear-shaped bottle solution at 200uL/min, hydrating for 10min at the temperature of 30 ℃ at 700r/min, cooling to room temperature, and then passing through a microfiltration membrane with the aperture of 450nm to obtain the thymosin beta-4 ethosome.
The particle size and PDI of the ethosome prepared in example 6 were measured, and the stability of the ethosome was measured by the standing method. Measured and observed, the results of examples 4, 5 and 6 are compared as shown in the following table:
example 7 investigation of physicochemical Properties of ethosomes
7.1 form
And (3) putting a proper amount of PRO liposome into a penicillin bottle, and observing that the appearance of the PRO liposome presents light blue opalescence. And taking one to two drops of the ethosome solution, diluting the ethosome solution by distilled water, dropping the diluted ethosome solution on a copper sheet, dyeing the copper sheet by using a 2% phosphotungstic acid solution, drying the copper sheet for 30min, and observing the microscopic morphology by a transmission electron microscope. As shown in figure 6, the ethosome under TEM is round in shape, smooth in surface without adhesion, uniform in particle size distribution, and obvious fingerprint-like patterns can be observed, and the ethosome is a large single-chamber ethosome with a large middle hydrophilic cavity and suitable for encapsulating hydrophilic drugs.
7.2 particle size and potential
Taking a proper amount of the prepared liposome solution, diluting by a proper time, and measuring by a dynamic light scattering instrument and a Malvern laser particle size analyzer to obtain a particle size of 270 +/-3 nm; the zeta potential is stabilized at-58 + -5 mv.
7.3 examination of stability of ethosome Placement
and (3) analysis: the ethosome is placed for 10 days and 20 days. The particle size and PDI are not obviously changed, and the stability is good. 7.4 transdermal Performance Studies of beta-4 Alosomes
Rat dorsal skin was sandwiched between diffusion cell lid (donor) and diffusion cell (recipient), the inner surface of the skin was immersed in isotonic solution physiological saline, 2mL β -4 ethosome solution and free β -4 drug were immersed in 5mL physiological saline for transdermal diffusion, respectively. 0.4mL of the solution was taken from the absorption cell at 1h, 2h, 4h, 6h, 8h, 10h, and 12h, respectively, and an equal amount of physiological saline was added. The enzyme-linked immunosorbent assay was used to measure the cumulative permeation (Q) per unit area of the two.
The experimental results are as follows:
and (3) analysis: compared with free medicines with equal concentration, the beta-4 ethosome has larger cumulative permeation amount per unit area and better transdermal performance.
Example 8 ethosome face mask
The gel is used as an important external preparation form, is widely applied to skin administration, and can increase the retention of the medicine on the skin surface. The gel comprises aqueous and oily gels, and the ethosome is an aqueous liquid, so the gel-carbomer with aqueous property is selected as the gel matrix of the liposome. The gel has the advantages of good spreadability, no greasiness, good adhesion to skin, no irritation, easy cleaning and the like, and is widely applied to gels of medicines and cosmetics.
8.1 preparation method
(1) Preparation of ethosome: preparing ethosome according to the method;
(2) putting 0.5g of carbomer gel 934 and 940 into 24.5g of distilled water, magnetically stirring at 400rpm/min for 3.5h, standing overnight at room temperature after stirring, fully swelling, slowly adding 0.125g of freeze-drying protective agent (sucrose, glucose or mannitol), and stirring to obtain colorless transparent blank gel matrix. Slowly adding the prepared ethosome solution into the blank gel matrix, and grinding and uniformly mixing to obtain the beta-4 ethosome gel.
Taking the beta-4 ethosome in the embodiment 4, adding 0.5g of carbomer gel 934 and 940, placing the mixture in 24.5g of distilled water, magnetically stirring the mixture at 400rpm/min for 3.5h, standing the mixture at room temperature after stirring for overnight, fully swelling the mixture, slowly adding 0.125g of freeze-drying protective agent (sucrose, glucose and mannitol) and uniformly stirring the mixture to obtain a colorless and transparent blank gel matrix. Slowly adding the prepared ethosome solution into the blank gel matrix, and grinding and uniformly mixing to obtain the beta-4 ethosome gel.
As shown in FIG. 7, the prepared beta-4 ethosome gel has uniform and consistent color and luster, is semitransparent, and has neutral pH value. The composition is applied to the back skin of rabbits for 4h, and is visually observed 30min, 1h, 24h and 72h after the medicine is removed, so that the back skin of the rabbits does not cause red swelling or macule and other adverse reactions.
8.2 appearance Properties
The prepared beta-4 ethosome gel has uniform and consistent color and luster, is semitransparent, and has neutral pH value. The composition is applied to the back skin of rabbits for 4h, and is visually observed 30min, 1h, 24h and 72h after the medicine is removed, so that the back skin of the rabbits does not cause red swelling or macule and other adverse reactions.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.
Claims (4)
1. The thymosin beta-4 ethosome is characterized by being prepared from the following raw materials in parts by weight: thymosin beta-40.022-0.043%, phospholipid 0.054-0.084%, cholesterol 0.018-0.028%, anionic surfactant 0.02-0.04%, ethanol 10.10-30.30% and the balance of distilled water;
the preparation method of the thymosin beta-4 ethosome comprises the following steps:
(1) dissolving phospholipid and cholesterol in absolute ethyl alcohol to obtain an alcohol phase solution, wherein the phospholipid is soybean phospholipid or lecithin;
(2) dissolving an anionic surfactant and thymosin beta-4 in distilled water to prepare an aqueous solution, wherein the anionic surfactant is sodium deoxycholate; slowly injecting the water phase solution into the alcohol phase solution under the stirring state for hydration;
(3) cooling the hydrated solution to room temperature, and filtering the solution through a microporous filter membrane to obtain the thymosin beta-4 ethosome, wherein the pore diameter of the microporous filter membrane is 450 nm;
the concentration of the ethanol solution in the mixed solution obtained in the step (3) is 25-40%; the stirring speed is 500-800 r/min; the injection rate of the water phase is 0.2 mL/min-2 mL/min; the hydration time is 10-30 min, and the hydration temperature is 25-40 ℃.
2. The thymosin beta-4 ethosome of claim 1, wherein the mass fraction ratio of the raw materials is: thymosin beta-40.03198, phospholipid 0.06396, cholesterol 0.02131, anionic surfactant 0.02665%, ethanol 25.23% and the balance of distilled water.
3. The thymosin beta-4 ethosome of claim 1, wherein said phospholipid is soy phospholipid.
4. Use of the thymosin beta-4 ethosome of any of claims 1 to 3 for the preparation of a repair mask.
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| CN201810951630.5A CN108721604B (en) | 2018-08-21 | 2018-08-21 | Thymosin beta-4 ethosome and preparation process thereof |
| PCT/CN2018/108526 WO2020037783A1 (en) | 2018-08-21 | 2018-09-29 | THYMOSIN β-4 ETHOSOME AND PREPARATION PROCESS THEREOF |
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| CN108721604A (en) | 2018-11-02 |
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