CN110141657B

CN110141657B - Epidermal growth factor liposome and preparation method thereof

Info

Publication number: CN110141657B
Application number: CN201910414446.1A
Authority: CN
Inventors: 寇龙发; 陈瑞杰; 姚情; 鲍仕慧; 孙锐; 肖姝艺
Original assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Current assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2023-01-17
Anticipated expiration: 2039-05-17
Also published as: CN110141657A

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses an epidermal growth factor liposome and a preparation method thereof. Soluplus and silk fibroin are utilized to synergistically improve the encapsulation efficiency and stability of the epidermal growth factor, and a reverse evaporation method is adopted to prepare the epidermal growth factor composite liposome. Compared with the prior art, the invention has the advantages of simple operation, safety, no toxicity, small irritation to skin, mild property and stable effect.

Description

Epidermal growth factor liposome and preparation method thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations or cosmetics, belongs to the technical field of biological materials, and particularly relates to an epidermal growth factor liposome and a preparation method thereof.

Background

The epidermal growth factor is a heat-resistant single-chain low-molecular polypeptide consisting of 53 amino acid residues, and can promote DNA synthesis and mitosis of target cells. Research shows that the epidermal growth factor can be used for treating various skin-related diseases, including diabetic foot, scald, eye cornea injury, dermatitis and the like, and realizes skin repair or regeneration. In addition, the epidermal growth factor can promote the proliferation and growth of epidermal cells, has the functions of resisting senility, protecting skin, protecting health and the like, and can be used as an additive of a cosmetic. However, the epidermal growth factor is a macromolecular active substance and has the defects of poor stability, low drug loading efficiency, low skin permeability and the like.

Liposomes are molecular assemblies composed of phospholipid bilayers, and have a closed vesicle structure similar to a biological membrane. On one hand, the special bilayer structure can be used for entrapping hydrophilic and hydrophobic active factors; on the other hand, the bionic structure of the biological membrane makes the biological membrane have good biocompatibility and safety. The liposome has good skin tolerance and good permeability, and the transdermal depth and the skin retention of the drug-encapsulated can be effectively increased through the reasonable design of the film-forming material. For example, the ethanol with higher concentration is used for replacing cholesterol in the liposome, so that the liposome with smaller particle size can be obtained, and the liposome has good flexibility and can promote the transdermal absorption of the medicament. However, liposomes, particularly when added with low molecular weight alcohols such as ethanol and propylene glycol, have poor stability, particularly in the form of liquid pharmaceutical preparations or cosmetic products.

Soluplus is white to yellowish round particles, has a chemical structure of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, is a novel polyethylene nonionic surfactant developed and marketed in 2009 by Pasteur Germany, and is an ideal solid dispersion preparation material. Reports show that the Soluplus can promote the gastrointestinal absorption of the medicine and greatly improve the oral bioavailability of the medicine. In addition, soluplus can be self-assembled to form a nano-micelle structure and is used as a nano delivery carrier of hydrophobic drugs. At present, no report related to the application of Soluplus as a transdermal absorption enhancer of a biological macromolecular medicament is found.

The silk fibroin is a natural high-molecular fiber protein, has good biocompatibility and low immunity, has nontoxic degradation products, can be absorbed by skin, and has excellent mechanical properties. The silk fibroin has special hydrophobic crystal region, and can fix bioactive compounds such as enzyme, antibody, growth factor, etc. However, silk fibroin has poor stability and is easy to mildew and deteriorate during storage and placement, thereby causing inactivation of the drug. At present, no relevant report on the synergistic improvement of the epidermal growth factor stability by Soluplus and silk fibroin exists.

At present, there is still a need for liposomes that can improve the storage stability of epidermal growth factor to address the limitations of epidermal growth factor use.

Disclosure of Invention

The technical problem to be solved by the embodiments of the present invention is to provide an epidermal growth factor liposome and a preparation method thereof. The epidermal growth factor liposome is simple to operate, safe, non-toxic, low in skin irritation, mild in property and stable in effect. The liposome prepared by the method has good stability, simple operation and good repeatability.

In order to achieve the above objects, a first aspect of the present invention is to provide an epidermal growth factor liposome

An epidermal growth factor liposome comprises epidermal growth factor, soluplus, silk fibroin and liposome film-forming material.

Further, the mass ratio of the epidermal growth factor, soluplus, silk fibroin and liposome lipid film-forming material is 1 (20-800): (5-200): (50-1000).

Furthermore, the particle size of the liposome is 50-300 nm.

Further, the lipid film-forming material comprises phospholipid and any one or combination of sterol and low molecular alcohol. The lipid film-forming material comprises essential phospholipid, and one or more of sterol and low molecular alcohol

Furthermore, the phospholipid is one or more of soybean lecithin, egg yolk lecithin, phosphatidylcholine, phosphatidylserine and polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine, preferably soybean lecithin and egg yolk lecithin; the sterol is preferably cholesterol; the low molecular alcohol is one or more of ethanol and propylene glycol.

Preferably, the composition is, based on 100mL nanoliposomes:

epidermal growth factor: 0.2-2 mg

Soluplus：5～200mg

Silk fibroin: 5-100 mg

Liposome film-forming material: 500-1500 mg

The invention also provides a method for preparing the epidermal growth factor liposome based on a reverse evaporation method, which comprises the following steps:

(1) Dissolving epidermal growth factor, soluplus and silk fibroin in water to form a water phase;

(2) Mixing the liposome film-forming material with an organic solvent to form an oil phase;

(3) Mixing the oil phase and the water phase, and performing ultrasonic treatment to obtain a white suspension;

(4) Rotary evaporating to remove organic solvent to form white gel substance;

(5) Adding appropriate amount of water, rotating continuously to dissolve the membrane and fully hydrate.

Further, the organic solvent is one or more of 1-4 halogenated alkanes, preferably dichloromethane and chloroform, and more preferably chloroform.

The application of the epidermal growth factor liposome in the preparation of medicines or cosmetics with skin improvement and repair effects is also within the protection scope of the invention.

Has the advantages that:

(1) The invention firstly proposes that the epidermal growth factor, soluplus and silk fibroin are compounded and wrapped in the nano liposome carrier, and the provided liposome structure wraps the three components, thereby avoiding the influence of the external environment on the epidermal growth factor, improving the placing stability, being convenient to use and having good product compatibility.

(2) The lipid materials lecithin and cholesterol in the lipid carrier have good physiological compatibility with skin, and the particle size is small (about 50nm-300 nm), so that epidermal growth factors and silk fibroin can be transferred into the skin through mechanisms such as fusion with the stratum corneum and penetration, and the epidermal growth factors and the silk fibroin can effectively play a role in repairing skin cells and wound healing.

(3) The epidermal growth factor liposome has good skin moisturizing, skin cell activating and skin conditioning effects, can be directly used as a skin care product or a medicament, is convenient and quick to apply, and has wide application prospects in the fields of cosmetics and medical materials.

(4) The epidermal growth factor liposome prepared by compounding the epidermal growth factor, soluplus and silk fibroin effectively improves the proliferation activity of crude cells, has the gain effect of skin repair, greatly improves the transdermal property and the skin absorption rate of active ingredients, and has stable performance and good use compatibility.

(5) The liposome prepared by the preparation method provided by the invention has the advantages of high encapsulation efficiency, simple operation, good repeatability and high stability.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for a person skilled in the art to obtain other drawings based on the drawings without paying creative efforts.

FIG. 1 shows the results of experiments on the cell proliferation promoting activity of the EGF liposome.

FIG. 2 is the experimental result of the effect of epidermal growth factor liposome in promoting diabetic wound healing.

Detailed Description

To make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1: preparation of epidermal growth factor liposome

According to Table 1, 2mg of epidermal growth factor (hEGF), soluplus, fibroin, and water 5 mL; mixing the liposome film-forming material with the prescription amount with 20mL of organic solvent to form an oil phase; mixing the oil phase and the water phase, and performing ultrasonic treatment to obtain a white suspension; rotary evaporating to remove organic solvent to form white gel substance; adding 75mL water, rotating continuously to dissolve the membrane and fully hydrate, and supplementing water to 100mL to obtain the epidermal growth factor liposome.

TABLE 1 epidermal growth factor liposome formulation

Note that the components indicating the indicated doses are replaced by the components in parentheses.

Example 2: particle size and surface potential of liposomes

The epidermal growth factor liposome prepared in example 1 was taken, and the particle size and encapsulation efficiency thereof were measured, respectively, and the results are shown in table 2. The epidermal growth factor liposome prepared in example 1 was stored at room temperature for 3 months, and then the particle size and encapsulation efficiency were measured, respectively, and the results are shown in table 2.

TABLE 2 particle size and encapsulation efficiency of epidermal growth factor liposomes

Serial number	Particle size (nm)	Encapsulation efficiency (%)	Particle size after storage (nm)	Encapsulation Rate (%)
					1	147.61	56.34	150.38	49.72
2	99.49	45.58	117.32	38.38
					3	159.35	20.17	166.73	17.38
4	235.67	37.42	410.63	23.42
					5	125.39	17.32	133.84	9.12
6	129.47	-	173.78	-
					7	162.42	15.94	183.37	8.84
8	125.39	30.28	136.74	28.71
					9	136.30	48.32	149.87	39.44
10	102.80	25.11	114.23	10.39
					11	105.47	30.44	154.43	25.42
12	137.61	42.30	166.45	17.43

The epidermal growth factor liposome has the advantages of spherical appearance, flat and smooth edge, normal particle size distribution and no significant difference in liposome shape in each experimental group, and the experimental preparation method is reasonable and stable under the condition of a suitable liposome film-forming material and can successfully prepare the liposome. However, the particle size of the liposome is significantly different from each group, and when propylene glycol is added into the membrane forming material, the particle size is significantly smaller than that of the group without the addition. Liposomes of small particle size will aid transdermal absorption of the drug. In the formula proportion range (formula 1 &2) of the invention, liposomes between 50 and 200nm can be successfully prepared according to the provided preparation method, and the liposome has better encapsulation efficiency (> 40%).

The epidermal growth factor liposome (formula 1 &2) provided by the invention has good storage stability, still maintains good nano size after being placed at room temperature for 3 months, and avoids the advanced leakage of active drugs.

Example 3 epidermal growth factor liposomes promoting cell proliferation Activity

The epidermal growth factor liposomes prepared in example 1 were collected, and the 3T3 cell proliferation promoting activity of each liposome group was examined by the MTT method. The epidermal growth factor liposomes prepared in example 1 were stored at room temperature for 3 months, and then the proliferation promoting activity of 3T3 cells by the liposomes of each group was examined by the MTT method.

The specific experimental process is as follows: 3T3 cells were plated at 1X 10 ⁶ Each well was inoculated in a 12-well culture plate, 50. Mu.L of each liposome solution was added to 6 wells of the plate, and a blank control group was set. After adding 100. Mu.L of MTT reagent at 48 hours and incubating at 37 ℃ for 4 hours, 150. Mu.L of DMSO was added to dissolve formazan precipitate, and the absorbance of the culture solution at a wavelength of 570nm was measured by a microplate reader to calculate the cell proliferation rate, which was calculated as:

the effect of liposomes in each group on cell proliferation activity is shown in FIG. 1. The epidermal growth factor liposome can promote the proliferation of 3T3 cells and has good storage stability.

Example 6: in vitro skin permeability study of epidermal growth factor liposomes

The epidermal growth factor liposome prepared in example 1 was taken and examined for skin permeability using a modified Franz drug transdermal diffusion tester. The specific experimental process is as follows: the prepared Wistar rat abdominal skin is fixed to a diffusion pool, 3mL of each liposome group is respectively taken out to a supply chamber, and the supply chamber is sealed by a preservative film to prevent water evaporation. 0.4mL of the receiving solution was sampled at 2,4,12h, respectively, while the same volume of fresh receiving solution at the same temperature was replenished. After the sample is centrifuged, the supernatant is taken and the content of the epidermal growth factor in the supernatant is determined by Elisa. The research finds that each group of epidermal growth factor liposome has good in-vitro skin permeability, and compared with the epidermal growth factor solution group, the skin drug storage amount is obviously improved. The in vitro skin permeability of each group of epidermal growth factor liposome has positive correlation with the encapsulation efficiency. The epidermal growth factor liposome (prescription 1 &2) of the present invention has the best in vitro rat skin permeability in each experimental group.

Example 7: epidermal growth factor liposome for promoting diabetic wound healing

The epidermal growth factor liposome prepared in example 1 was taken and examined for its effect of promoting wound healing in diabetes. The specific experimental process is as follows: taking a model diabetic rat, and removing the full-thickness skin with the area of about 1cm multiplied by 1cm from the dorsal side to form a wound surface. After random grouping, 200 mu L of each group of liposome is smeared on the wound surface, 3 times a day; the blank control group was smeared with an equal amount of physiological saline. Recording the size of the wound by using a transparent film marking method on 7 and 14 days after the wound respectively, and calculating the wound healing rate by the following formula:

the wound repair effect of the liposomes in each group on diabetic rats is shown in figure 2. The epidermal growth factor liposome can promote wound repair of diabetic SD rats.

Example 9: study on skin application effect of epidermal growth factor liposome

Trial observation is carried out on the skin moistening cream formed by mixing the epidermal growth factor-containing liposome prepared by the method with an emulsion matrix. Healthy women aged 30-45 years are selected as subjects, and the faces of the subjects show dark yellow and color spots to different degrees. Each group had 20 persons, and 3 subjects of the group used a skin lotion prepared by mixing liposomes with an emulsion base (5, 95, v/v) according to example 1, and applying the lotion to the test area (same area) washed with clear water once a day in the morning and at night. The continuous use for 28 days is a treatment course. The curative effect standard is as follows: (1) effect-showing: the dark yellow or the color spot area is faded by more than 80 percent, and the skin is obviously smooth and moist; (2) effective: the dark yellow or the color spot area is reduced by more than 30 percent, and the skin is smooth and moist; and (3) invalidation: there was no change before and after treatment. The results are shown in Table 3:

TABLE 3 skin lotion made from EGF liposome

Serial number	Show effect	Is effective	Invalidation	Total effective rate (%)
					1	10	8	2	90
2	15	4	1	95
					3	0	3	17	15
4	2	5	13	35
					5	2	3	15	25
6	1	2	17	15
					7	0	4	16	20
8	0	2	18	10
					9	3	7	10	50
10	1	2	17	15
					11	1	5	14	30
12	2	7	11	45

The experimental results show that the epidermal growth factor liposome can obviously improve the problem of pigmentation on the surface of female skin. The epidermal growth factor compound liposome is added into cosmetics as a multifunctional powerful cell metabolism promoting factor, has obvious skin care effect, has the effects of smoothing, moistening, whitening, moisturizing, resisting aging, repairing damaged skin, removing freckles, removing acnes and the like, and provides complete protection for the skin.

In addition, the invention can also be applied to cosmetics which comprise the epidermal growth factor liposome, and the types of the cosmetics comprise skin-care products, anti-wrinkle products, cleaning cosmetics, functional cosmetics and beauty cosmetics; the cleaning cosmetics comprise cleaning cream, face washing cream, bath foam and shaving products; the functional cosmetics comprise whitening skin care products, anti-wrinkle skin care products, moisturizing skin care products, acne-removing skin care products and anti-sweat skin care products; the cosmetics comprise foundation cream, face powder, face mask, lipstick and popliteal.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. An epidermal growth factor liposome, comprising: the epidermal growth factor, soluplus and silk fibroin are compounded and wrapped in a nano liposome carrier, wherein the chemical structure of the Soluplus is a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and the nano liposome carrier is formed by a liposome film-forming material; the particle size of the liposome is 50 to 300nm;

based on 100mL of liposome, the liposome consists of,

epidermal growth factor 2mg

Soluplus 75mg

Silk fibroin 150mg

1200mg of soybean lecithin serving as a liposome film-forming material and 100mg of cholesterol;

or epidermal growth factor 2mg

Soluplus 125mg

Silk fibroin 275mg

The liposome film forming material comprises 1000mg of yolk lecithin, 80mg of cholesterol and 100mg of propylene glycol.

2. A method for preparing the epidermal growth factor liposome of claim 1 based on reverse evaporation method, characterized by comprising the following steps:

(4) Rotary evaporation to remove the organic solvent and form a white gel;

(5) Adding a proper amount of water, continuously rotating to dissolve and fully hydrate the membrane, and obtaining the epidermal growth factor liposome.

3. The method of claim 2, wherein: the organic solvent is one or more of 1-4 halogenated alkanes.

4. The use of the epidermal growth factor liposome based on the claim 1 in the preparation of cosmetics comprising the epidermal growth factor liposome, and the cosmetic categories comprise cleaning cosmetics, functional cosmetics and cosmetology cosmetics; the cleaning cosmetics comprise cleaning cream, face washing cream, bath foam and shaving products; the functional cosmetics comprise whitening skin care products, anti-wrinkle skin care products, moisturizing skin care products, acne-removing skin care products and anti-sweat skin care products; the beauty cosmetics comprise foundation cream, fragrant powder, facial masks, lipstick and rouge.