CN110141657B - A kind of epidermal growth factor liposome and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and specifically discloses an epidermal growth factor liposome and a preparation method thereof. The epidermal growth factor liposome comprises epidermal growth factor, Soluplus, silk fibroin and a liposome film-forming material. Soluplus and silk fibroin were used to synergistically improve the encapsulation efficiency and stability of epidermal growth factor, and the reverse evaporation method was used to prepare complex liposomes of epidermal growth factor. Compared with the prior art, the invention is simple in operation, safe and non-toxic, less irritating to the skin, mild in nature and stable in effect.

Description

A kind of epidermal growth factor liposome and preparation method thereof

technical field

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

Background technique

Epidermal growth factor is a heat-resistant single-chain low-molecular-weight polypeptide composed of 53 amino acid residues, which can promote DNA synthesis and mitosis of target cells. Studies have shown that epidermal growth factor can be used for a variety of skin-related diseases, including diabetic foot, scald, corneal injury, dermatitis, etc., to achieve skin repair or regeneration. In addition, epidermal growth factor can promote the proliferation and growth of epidermal cells, and can also play anti-aging and skin care functions, and can be used as an additive for cosmetic products. However, EGF is a macromolecular active substance, which has defects such as poor stability, low drug loading efficiency, and low skin permeability.

Liposomes are molecular assemblies composed of phospholipid bilayers, which have a closed vesicle structure similar to biological membranes. On the one hand, its special bilayer structure can be used to entrap hydrophilic and hydrophobic active factors; on the other hand, its biomimetic structure makes it have good biocompatibility and safety. Liposomes have good skin tolerance and good permeability. Through reasonable design of film-forming materials, the transdermal depth and skin retention of entrapped drugs can be effectively increased. For example, replacing cholesterol in liposomes with higher concentration of ethanol can obtain liposomes with smaller particle size, good flexibility, and promote drug transdermal absorption. However, liposomes, especially after adding low-molecular alcohols such as ethanol and propylene glycol, have poor stability, especially pharmaceutical preparations or beauty products that exist in liquid form.

Soluplus is white to yellowish round particles, the chemical structure is polyethylene caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, it is a new type of polyethylene non-ionic surfactant developed by BASF in 2009. It is an ideal solid dispersion preparation material. Reports have shown that Soluplus can promote the gastrointestinal absorption of drugs and greatly improve the oral bioavailability of drugs. In addition, Soluplus can self-assemble to form nano-micelle structure, which is used as a nano-delivery carrier for hydrophobic drugs. At present, there are no relevant reports about Soluplus as a transdermal absorption accelerator for biomacromolecular drugs.

Silk fibroin is a natural polymer fibrin with good biocompatibility, low immunity, non-toxic degradation products, can be absorbed by the skin and excellent mechanical properties. Silk fibroin has a special hydrophobic crystalline region, which can immobilize biologically active compounds, such as enzymes, antibodies, growth factors, etc. However, silk fibroin has poor stability and is prone to mildew and deterioration during storage, thereby causing drug inactivation. At present, there is no relevant report about the synergistic improvement of the stability of epidermal growth factor by Soluplus and silk fibroin.

At present, there is still a need for liposomes capable of improving the storage stability of EGF to address the limitations of EGF use.

Contents of the 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 of the invention is simple to operate, safe and non-toxic, less irritating to the skin, mild in nature and stable in effect. According to the method, the liposome prepared by the method has good stability, simple operation and good repeatability.

To achieve the above object, the first technical scheme of the present invention provides a kind of epidermal growth factor liposome

A kind of 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).

Further, the particle diameter of the liposome is 50-300 nm.

Further, the lipid membrane-forming material includes any one or several combinations of phospholipids, sterols, and low-molecular alcohols. That is, the lipid film-forming material includes necessary phospholipids, and any one or several combinations of sterols and low-molecular alcohols

Further, the phospholipid is one or more of soybean lecithin, egg yolk lecithin, phosphatidylcholine, phosphatidylserine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, preferably soybean egg Phospholipids, egg yolk lecithin; the sterol is preferably cholesterol; the low molecular weight alcohol is one or more of ethanol and propylene glycol.

Preferably, in terms of 100mL nano-liposomes, it consists of:

Epidermal growth factor: 0.2～2mg

Soluplus: 5~200mg

Silk fibroin: 5~100mg

Liposome film-forming material: 500～1500mg

The present invention also provides a method for preparing epidermal growth factor liposomes based on the reverse evaporation method, comprising the steps of:

(1) Dissolving epidermal growth factor, Soluplus, and silk fibroin in water to form an aqueous phase;

(2) liposome membrane-forming material is mixed with an organic solvent to form an oil phase;

(3) mixing the oil phase and the water phase, and ultrasonically obtaining a white suspension;

(4) rotary evaporation removes organic solvent, forms white gel substance;

(5) Add an appropriate amount of water and continue to rotate to dissolve the film and fully hydrate it.

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

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

Beneficial effect:

(1) The present invention proposes for the first time that epidermal growth factor, Soluplus, and silk fibroin are compounded and wrapped in nanoliposome carrier, and the liposome structure provided wraps the three components in it, avoiding the impact of external environment on epidermal growth factor Impact, improve placement stability, easy to use, good product compatibility.

(2) The lipid materials lecithin and cholesterol in the lipid carrier have good physiological compatibility with the skin, and the particle size is small (about 50nm-300nm), which can be merged with the stratum corneum, penetrated and other mechanisms , to transmit epidermal growth factor and silk fibroin into the skin, so that epidermal growth factor and silk fibroin can effectively repair skin cells and wound healing.

(3) The epidermal growth factor liposome itself has good skin moisturizing, activating skin cells and skin conditioning effects, and can be directly used as skin care or medicine, with convenient application and broad application prospects in the fields of cosmetics and medical materials.

(4) Epidermal growth factor liposome prepared by compounding epidermal growth factor, Soluplus, and silk fibroin can effectively improve the proliferative activity of rough cells, have the effect of gaining skin repair, and greatly improve the transdermal property of active ingredients , skin absorption rate, and stable performance, good compatibility.

(5) The liposomes prepared by the preparation method provided by the invention have high encapsulation efficiency, simple and convenient operation, good repeatability and high stability.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, obtaining other drawings based on these drawings still belongs to the scope of the present invention without any creative effort.

Fig. 1 is the experimental result of epidermal growth factor liposome promoting cell proliferation activity.

Fig. 2 is the experimental result of epidermal growth factor liposome promoting diabetic wound healing.

detailed description

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings.

Embodiment 1: the preparation of epidermal growth factor liposome

According to Table 1, 2mg epidermal growth factor (hEGF), prescription quantity Soluplus, prescription quantity silk fibroin are dissolved in 5mL water; The prescription quantity liposome film-forming material is mixed with 20mL organic solvent to form an oil phase; Oil phase and water Mix with each other and ultrasonically obtain a white suspension; remove the organic solvent by rotary evaporation to form a white gel-like substance; add 75mL of water and continue to rotate to dissolve and fully hydrate the film, and make up water to 100mL to obtain the epidermal growth factor liposome.

Table 1 Epidermal growth factor liposome prescription

Note: * indicates that the ingredients in the marked dosage are replaced by the ingredients in brackets.

Embodiment 2: particle size and surface potential of liposome

Get the epidermal growth factor liposome prepared in embodiment 1, measure its particle size and encapsulation efficiency respectively, the results are shown in Table 2. Get the epidermal growth factor liposome prepared in embodiment 1, after placing 3 months under room temperature, measure its particle size and encapsulation efficiency respectively, the results are shown in Table 2.

Particle size and encapsulation efficiency of table 2 epidermal growth factor liposome

serial number Particle size (nm) Encapsulation rate (%) Particle size after storage (nm) Encapsulation rate after storage (%) 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 appearance form of the present invention is spherical, and the edge is flat and smooth, and particle diameter is normal distribution substantially, and the liposome shape in each experimental group has no significant difference, shows that in suitable liposome film-forming material Under the conditions, the preparation method of this experiment is relatively reasonable and stable, and liposomes can be successfully prepared. But there is significant difference between each group of the particle diameter of liposome, when adding propylene glycol in the film-forming material, its particle diameter is all significantly less than not adding group. Liposomes with small particle size will facilitate the transdermal absorption of drugs. Within the range of the formulation ratio of the present invention (prescription 1 & 2), liposomes with a diameter of 50-200 nm can be successfully prepared according to the preparation method provided, and have better encapsulation efficiency (>40%).

The epidermal growth factor liposome (prescription 1&2) of the present invention has good storage stability, and after being placed at room temperature for 3 months, it still maintains a good nanometer size, and avoids the early leakage of active drugs.

Example 3. Epidermal growth factor liposome promotes cell proliferation activity

Get the epidermal growth factor liposome that prepares in embodiment 1, utilize MTT method to detect each group liposome promotes 3T3 cell proliferation activity. Get the epidermal growth factor liposome that prepares in embodiment 1, after placing 3 months under room temperature condition, utilize MTT method to detect each group liposome promotes 3T3 cell proliferation activity.

The specific experimental process is as follows: 3T3 cells were inoculated in 12-well culture plate at 1×10 ⁶ per well, 50 μL of liposome solution of each group was added to the cell solution in 6 wells, and a blank control group was set. At 48 hours, add 100 μL MTT reagent and incubate at 37°C for 4 hours, then add 150 μL DMSO to dissolve the formazan precipitate, measure the absorbance value of the culture solution at a wavelength of 570 nm with a microplate reader, and calculate the cell proliferation rate. The calculation formula is:

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

Embodiment 6: the in vitro skin permeability investigation of epidermal growth factor liposome

Get the epidermal growth factor liposome prepared in embodiment 1, adopt improved Franz drug transdermal diffusion tester to investigate its skin permeability. The specific experimental process is as follows: use the prepared abdominal skin of Wistar rats to fix to the diffusion cell, take 3mL of liposomes from each group into the supply chamber, and seal the supply chamber with plastic wrap to prevent water evaporation. Sampling 0.4mL from the receiving solution at 2, 4, and 12 hours respectively, and supplementing the same volume of fresh receiving solution at the same temperature at the same time. After the sample was centrifuged, the supernatant was taken to measure the content of epidermal growth factor in it by Elisa. The study found that the epidermal growth factor liposomes in each group had good in vitro skin permeability, and compared with the epidermal growth factor solution group, the drug retention in the skin was significantly improved. The in vitro skin permeability of each group of EGF liposomes had a positive correlation with their encapsulation efficiency. Epidermal growth factor liposomes of the present invention (prescription 1&2) have the best in vitro rat skin permeability in each experimental group.

Embodiment 7: epidermal growth factor liposome promotes wound healing effect of diabetes

The epidermal growth factor liposome prepared in Example 1 was taken to investigate its effect on promoting diabetic wound healing. The specific experimental process is as follows: take model diabetic rats, and remove the full-thickness skin with an area of about 1 cm × 1 cm on the back side to form a wound. After random grouping, 200 μL of liposomes of each group were applied to the wound surface, 3 times a day; the same amount of normal saline was applied to the blank control group. On the 7th and 14th day after injury, the wound size was recorded by the transparent film marking method, and the wound healing rate was calculated. The calculation formula is:

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

Embodiment 9: Research on skin application effect of epidermal growth factor liposome

The skin lotion formed by mixing the liposome containing epidermal growth factor prepared above with the emulsion base was tested and observed. Healthy women aged 30-45 were selected as the subjects, and the faces of the subjects all showed different degrees of dark yellow and stains. Every group of 20 people, wherein 3 groups of subjects used the body lotion mixed with liposomes and emulsion matrix (5:95, v/v) in Example 1 respectively, in the test area (same area) washed with clear water. ) Apply lotion, once a day in the morning and evening. Continuous use for 28 days is a course of treatment. Efficacy criteria: (1) Marked effect: more than 80% of the dark yellow or pigmented area subsides, and the skin is obviously smooth and moisturized; (2) effective: more than 30% of the dark yellow or pigmented area subsides, and the skin is smoother and moisturized; (3) Ineffective: no change before and after treatment. The results are shown in Table 3:

Table 3 EGF liposome-prepared body lotion use effect

serial number markedly effective efficient invalid 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 above-mentioned experimental results show that the epidermal growth factor liposome of the present invention can significantly improve the problem of female skin surface pigmentation. The epidermal growth factor complex liposome of the present invention is added to cosmetics as a multifunctional and powerful cell metabolism promoting factor, which has a remarkable effect on skin care, and has the functions of smoothing, moisturizing, whitening, moisturizing, anti-aging, and repairing damaged skin. , anti-freckle, anti-acne and other effects, to provide comprehensive protection for the skin.

In addition, the present invention can also be applied in cosmetics, which include the epidermal growth factor liposome, and the cosmetics include skin care and anti-wrinkle products, cleaning cosmetics, functional cosmetics and beauty cosmetics; The cleaning cosmetics include cleansing cream, facial cleanser, body wash and shaving products; the functional cosmetics include whitening skin care products, anti-wrinkle skin care products, moisturizing skin care products, acne-removing skin care products and anti-perspiration skin care products; Beauty cosmetics include foundation cream, face powder, face mask, lipstick and rouge.

The above disclosures are only preferred embodiments of the present invention, and certainly cannot limit the scope of rights of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope of the present invention.

Claims (4)

1. an epidermal growth factor liposome is characterized in that comprising: by nano liposome carrier, epidermal growth factor, Soluplus, silk fibroin compound are wrapped in wherein, and the chemical structure of described Soluplus is polyethylene Caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, the nano-liposome carrier is formed by liposome membrane-forming material; the particle diameter of the liposome is 50 ~ 300nm; Based on 100mL liposome, its composition is, Epidermal Growth Factor 2mg Soluplus 75mg Silk Fibroin 150mg Liposome film-forming material soybean lecithin 1200mg, cholesterol 100mg; or epidermal growth factor 2mg Soluplus 125mg Silk Fibroin 275mg Liposome film-forming material egg yolk lecithin 1000mg, cholesterol 80mg, propylene glycol 100mg. 2. A method for preparing the epidermal growth factor liposome according to claim 1 based on reverse evaporation method, characterized in that it may further comprise the steps: (1) Dissolve epidermal growth factor, Soluplus, and silk fibroin in water to form an aqueous phase; (2) mixing the liposome membrane-forming material with an organic solvent to form an oil phase; (3) Mix the oil phase and the water phase, and ultrasonically obtain a white suspension; (4) Remove the organic solvent by rotary evaporation to form a white gel substance; (5) Add an appropriate amount of water and continue to rotate to dissolve the membrane and fully hydrate it to obtain the epidermal growth factor liposome. 3. The method according to claim 2, characterized in that: the organic solvent is one or more of 1-4 halogenated alkanes. 4. a kind of application based on the epidermal growth factor liposome described in claim 1 in being used to prepare cosmetics, including described epidermal growth factor liposome in this cosmetic, this cosmetic category comprises cleaning class cosmetics, functional Sexual cosmetics and beauty cosmetics; the cleansing cosmetics include cleansing cream, facial cleanser, body wash and shaving products; the functional cosmetics include whitening skin care products, anti-wrinkle skin care products, moisturizing skin care products, acne skin care products and Antiperspirant skin care products; said beauty cosmetics include foundation cream, face powder, facial mask, lipstick and rouge.

Images