CN109966170B - Flexible liposome cosmetic containing biological macromolecules and preparation method thereof - Google Patents
Flexible liposome cosmetic containing biological macromolecules and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of cosmetics, and particularly relates to a cosmetic containing flexible liposome of biomacromolecules and a preparation method thereof. The invention provides a cosmetic containing biomacromolecule flexible liposome aiming at the problems that most active ingredients in cosmetics containing biomacromolecule are difficult to permeate the skin due to the barrier effect of the cuticle of the skin epidermis, and the cosmetic is prepared by hydrophobic modified polypeptide DP7-C or PAL-DP7 modified flexible liposome containing biomacromolecule, so that the permeability of biomacromolecule in the cosmetic is effectively improved, a cosmetic which is easier to absorb and has better antioxidant and anti-aging effects is developed, and the application prospect is wide.
Description
Technical Field
The invention belongs to the field of cosmetics, and particularly relates to a flexible liposome cosmetic containing biomacromolecules and a preparation method thereof.
Background
In recent years, genetic engineering products are more and more widely applied to the field of beauty and skin care, and more than ten genetic engineering products are internationally used for slimming and shaping, skin repairing and damaging and the like. Biological macromolecules, especially cell growth factors, play an important role in the beauty and skin care, and the cell growth factors are originally present in skin cells, so that the skin metabolism is slowed down, the synthesis of the cell growth factors is reduced, and the skin gradually loses elasticity and vitality with the increase of age. The cell growth factor is produced by a DNA recombination technology, and is added into cosmetics at a certain effective concentration, so that the problems can be effectively solved, cells are activated, the collagen content is increased, the skin aging is delayed, and the skin is young, smooth and elastic.
The largest problem exists in the application of active ingredients of cell growth factors in beauty products, namely the transdermal absorption of macromolecular protein molecules. At present, cosmetics on the market are called to be added with various growth factors, nutritional ingredients and the like, the problem of skin absorption is not really solved, the nutritional factors only stay on the surface of the skin, the problem on the surface of the skin can be solved only temporarily, the nutritional factors cannot reach the deep layer of the skin directly, the cell activation level is fundamentally improved, and the skin aging is delayed.
Therefore, the development of cosmetics capable of carrying out efficient transdermal delivery on biomacromolecules, such as active substances with antioxidant and anti-aging effects like cell growth factors, has important significance.
Disclosure of Invention
The invention aims to provide a flexible liposome cosmetic containing biological macromolecules.
The invention provides a cosmetic containing flexible liposome of biological macromolecules, which is prepared by hydrophobic modified polypeptide modified flexible liposome containing biological macromolecule active ingredients.
In the flexible liposome cosmetic containing biological macromolecules, the hydrophobic modified polypeptide is a nitrogen-end hydrophobic modified polypeptide.
In the cosmetic containing the flexible liposome of the biological macromolecule, the flexible liposome modified by the hydrophobic modified polypeptide is prepared from the following raw materials in parts by weight: lecithin-sodium deoxycholate or polysorbate 80 derivative 7:2-4, 1-10 wt% of hydrophobic modified polypeptide, and biological macromolecule active ingredient.
Wherein, the cosmetic containing the flexible liposome of the biomacromolecule also contains an antioxidant. The antioxidant comprises: at least one of vitamin C or its derivative, vitamin E or its derivative, or coenzyme Q. The weight percentage of the antioxidant is 0.1-1%.
In the flexible liposome cosmetic containing the biological macromolecules, the biological macromolecules are growth factors or enzymes. In particular to at least one of fibroblast growth factor bFGF, epidermal growth factor EGF, keratinocyte growth factor KGF, vascular endothelial cell growth factor VEGF, superoxide dismutase SOD or lysozyme.
In the flexible liposome cosmetic containing the biological macromolecules, the weight percentage of the biological macromolecules is 1-10%.
The soft liposome cosmetic containing the biomacromolecule is prepared from the following components in parts by weight: lecithin-sodium deoxycholate or polysorbate 80 derivative 7:3, 0.5% by weight of antioxidant, and 2-10% by weight of hydrophobization modified polypeptide.
Wherein, in the flexible liposome cosmetic containing biological macromolecules, the sequence of the hydrophobic modified polypeptide is that NH is connected to the C end on the basis of SEQ ID NO. 12And coupling sterol compounds or saturated straight-chain fatty acids at the nitrogen terminal of the polypeptide.
1 amino acid sequence of hydrophobic modified polypeptide of SEQ ID NO
VQWRIRVAVIRK。
In the flexible liposome cosmetic containing biological macromolecules, the sterol compound is a cholesterol compound or a cholic acid compound.
In the flexible liposome cosmetic containing biological macromolecules, the sterol compound is at least one of cholesterol, succinylated cholesterol, cholic acid or deoxycholic acid.
In the flexible liposome cosmetic containing biological macromolecules, the saturated straight-chain fatty acid is at least one of C6-C20.
In the flexible liposome cosmetic containing biological macromolecules, the saturated straight-chain fatty acid is at least one of C8-C18.
In the flexible liposome cosmetic containing the biological macromolecules, the long-chain fatty acid is at least one of stearic acid, palmitic acid, lauric acid or n-caprylic acid.
In the flexible liposome cosmetic containing biological macromolecules, the hydrophobic modified polypeptide has the following structure:
Wherein, in the flexible liposome cosmetic containing biological macromolecules, R is
In the flexible liposome cosmetic containing biological macromolecules, the fibroblast growth factor bFGF in the specially modified flexible liposome accounts for 1-10% of the liposome by weight.
In the flexible liposome cosmetic containing biological macromolecules, the fibroblast growth factor bFGF in the specially modified flexible liposome accounts for 2-8% of the weight of the liposome.
In the flexible liposome cosmetic containing biological macromolecules, the fibroblast growth factor bFGF in the specially modified flexible liposome accounts for 5% of the weight of the liposome.
In the flexible liposome cosmetic containing biological macromolecules, the epidermal growth factor EGF in the specially modified flexible liposome accounts for 1-10% of the liposome by weight.
In the flexible liposome cosmetic containing biological macromolecules, the epidermal growth factor EGF in the specially modified flexible liposome accounts for 2-6% of the liposome by weight.
In the flexible liposome cosmetic containing biological macromolecules, the epidermal growth factor EGF in the specially modified flexible liposome accounts for 3% of the liposome by weight.
In the cosmetic containing the flexible liposome of the biomacromolecule, the weight percentage of superoxide dismutase SOD in the specially modified flexible liposome is 1-5% of the liposome.
In the cosmetic containing the flexible liposome of the biomacromolecule, the weight percentage of superoxide dismutase SOD in the specially modified flexible liposome is 1-3% of the liposome.
In the cosmetic containing the flexible liposome of the biomacromolecule, the superoxide dismutase SOD in the specially modified flexible liposome accounts for 2 percent of the weight of the liposome.
In the flexible liposome cosmetic containing the biological macromolecules, the flexible liposome cosmetic containing the biological macromolecules further comprises at least one of an emulsifier, a co-emulsifier, a skin conditioner, a whitening agent, a colorant, a humectant, a solubilizer, a surfactant, a preservative, a fragrance, an emollient, an anti-acne agent, a film forming agent, a thickener, a pH regulator, a buffer, a stabilizer or an ultraviolet absorbent which are related to the preparation of the cosmetic.
In the flexible liposome cosmetic containing the biological macromolecules, the dosage form of the flexible liposome cosmetic containing the biological macromolecules is water, emulsion, paste, jelly, lozenge or aerosol.
In the flexible liposome cosmetic containing the biological macromolecules, the flexible liposome cosmetic containing the biological macromolecules is in the form of lotion, essence, cream, facial mask, freeze-dried powder or facial cleanser.
The invention also provides a method for preparing the flexible liposome cosmetic containing the biomacromolecule, which comprises the following steps:
a. weighing lecithin, sodium deoxycholate or polysorbate 80 and vitamin E, placing into a reaction container, adding a solvent for dissolving, wherein the solvent is a mixture of chloroform and ethanol at a ratio of 1: 1-3 or is ethanol;
b. vacuum rotary evaporating to obtain liposome membrane, and drying;
c. adding DP7-C or PAL-DP7 and distilled water into the liposome membrane, and performing ultrasonic hydration to obtain blank liposome solution;
d. adding the bioactive macromolecules into the blank liposome solution, slightly shaking, and incubating at room temperature for 30-60 min to obtain the flexible liposome containing the bioactive macromolecules.
Wherein, the method for preparing the flexible liposome cosmetic containing the biomacromolecule further comprises the following steps of: extruding the incubated liposome to pass through a 0.1-0.45 mu m polycarbonate membrane for 4-8 times.
Wherein, the method for preparing the flexible liposome cosmetic containing the biomacromolecule further comprises the following steps of f: according to the dosage form requirement, the flexible liposome is further added with at least one of emulsifying agent, auxiliary emulsifying agent, skin conditioning agent, whitening agent, colorant, humectant, solubilizer, surfactant, antiseptic, aromatic, emollient, anti-acne agent, film forming agent, thickener, pH regulator, buffer, stabilizer or ultraviolet absorbent to prepare cosmetic dosage form.
The cosmetic is a daily chemical industrial product which is applied to any part (skin, hair, nails, lips and the like) of the surface of a human body by smearing, spraying or other similar methods so as to achieve the purposes of cleaning, eliminating bad smell, protecting skin, beautifying and decorating.
The flexible liposome is a self-aggregating vesicle improved by prescription on the basis of liposome, and a surface active substance such as sodium cholate, polysorbate 80 or derivatives thereof and the like is added into the phospholipid component of the liposome, so that the lipid membrane of the liposome has high deformability, and the liposome is also called a transfersome. The carrier has smaller particle size than liposome, and can pass through small holes with the pore size of 1/10-1/5, and the permeation rate and the permeation quantity are almost equal to those of pure water.
The action mechanism of the flexible liposome (transfersome) of the invention is as follows: 1. the particle size of the carrier is smaller than that of the liposome, and the carrier can quickly penetrate through the horny layer of the skin and enter the epidermis and dermis to form a reservoir; 2. the skin active ingredients and water encapsulated in the transfer body can be slowly released, and the action effect of the active substances is greatly improved. 3. The carrier carries encapsulated skin active substance cell growth factors, and utilizes the functions of the cell growth factors, such as repairing skin injury, promoting wound healing and stimulating collagen secretion, and repairing injury and scars caused by ultraviolet irradiation, environment and accidental injury;
the invention has the beneficial effects that:
the invention provides a novel flexible liposome based on the traditional liposome through prescription improvement, surface active substances such as sodium deoxycholate, polysorbate 80 or derivatives thereof and the like are added into phospholipid components of the liposome, so that a lipid membrane of the liposome has high deformability, and meanwhile, cell-penetrating peptide capable of enhancing transdermal effect is added, so that the transdermal permeability of the liposome is improved. The inventor uses the transfersome to wrap biological macromolecule, especially cell growth factor, uses the cell growth factor to repair skin injury, promote wound healing and stimulate the characteristic of collagen secretion, uses the transfersome to wrap the cell growth factor, uses its excellent transdermal capacity to prepare a novel cell growth factor transdermal agent, and develops its application in the cosmetic field. The novel cell growth factor transdermal agent is safe and nontoxic in used materials, and has good effects on repairing skin tissues, promoting collagen secretion, reducing wrinkle aggregation and melanin deposition. In addition, it has the functions and characteristics of biological membrane, strong affinity with human body cell, unique efficacy in cosmetics and great economic benefit.
On the technical level, the invention has two advantages: firstly, the product has the general functions of resisting aging, moisturizing and promoting softness in the market, and a novel transdermal technology is applied to cosmetics; secondly, the active substance encapsulated by the liposome is an active substance which is obtained by large-scale fermentation by utilizing a bioengineering technology and has special effect on skin nurturing, and fills the blank of a macromolecular transdermal technology of cosmetics in China.
Drawings
FIG. 1 is a nanometer particle size test chart of prepared bFGF-containing DP7-C modified flexible liposome with polysorbate 80 as a surfactant;
FIG. 2 is a nano-particle size test chart of prepared bFGF-containing PAL-DP7 modified flexible liposomes with polysorbate 80 as a surfactant;
FIG. 3 is a nanoparticle size test chart of EGF-containing DP7-C modified flexible liposomes prepared with polysorbate 80 as a surfactant;
FIG. 4 is a nano-particle size assay of EGF containing PAL-DP7 modified flexible liposomes prepared with polysorbate 80 as surfactant;
FIG. 5 is a nanometer particle size test chart of prepared SOD-containing DP7-C modified flexible liposome with polysorbate 80 as a surfactant;
FIG. 6 is a nano-particle size assay of prepared SOD-containing PAL-DP7 modified flexible liposomes using polysorbate 80 as a surfactant;
FIG. 7 is a graph showing the in vitro transdermal results of the prepared bFGF-containing DP7-C modified flexible liposome (1-1) using sodium deoxycholate as a surfactant: SDS-PAGE; western Blot;
FIG. 8 is a diagram showing the results of ELISA detection of the in vitro transdermal effect of the prepared bFGF-containing flexible liposomes;
FIG. 9 is a diagram showing the result of ELISA test for the in vitro transdermal effect of the prepared flexible liposome containing EGF;
FIG. 10 is a diagram showing the results of ELISA assay for the in vitro transdermal effect of the prepared flexible liposomes containing SOD;
FIG. 11 is a graph of the results of immunohistochemical assays: A. slicing the skin of a mouse not coated with the flexible liposome; B. mice skin sections smeared at the site of the flexible liposomes.
Detailed Description
The following examples further illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention to the examples.
The hydrophobic modified polypeptide DP7-C is a conjugate of antimicrobial peptide DP7 and cholesterol, the hydrophobic modified polypeptide PAL-DP7 is a conjugate of antimicrobial peptide DP7 and palmitic acid, the synthetic method is disclosed in patent CN107441501A, and other methods in the field can be adopted for synthesis, and DP7-C and PAL-DP7 used in the examples are synthesized by Dorkaee biological medicine science and technology development Co.
The raw materials and equipment used in the examples of the present invention are all common commercial products.
Example 1 preparation of Flexible liposomes containing biological macromolecules according to the invention
Precisely weighing soybean lecithin, sodium deoxycholate or polysorbate 80 or its derivative, and vitamin E according to the formulation, dissolving with chloroform/ethanol or ethanol respectively, dissolving and mixing in 250ml pear-shaped bottle, rotary evaporating at room temperature for 2hr, and placing the obtained film in a vacuum drying oven overnight. Distilled water and a proper amount of hydrophobic modified polypeptide are added in the next day, and a 400w probe is used for ultrasonic treatment for 30min to obtain a blank carrier solution. Then adding proper amount of bFGF (or EGF, SOD, etc.) slowly, incubating for 40min at room temperature, filtering the obtained mother liquor with 0.2 μm polycarbonate membrane for multiple times, adding excipient 5% mannitol, subpackaging in penicillin bottles, and lyophilizing to obtain the flexible liposome containing biological macromolecules of the invention.
Experimental example 1-1 preparation of DP7-C modified Flexible Liposome containing bFGF
Experimental materials:
soybean lecithin: purchased from Lipoid GmbH;
sodium deoxycholate: purchased from biotechnology responsibility ltd of obozoxing, beijing;
polysorbate 80: from Merck, USA;
hydrophobically modified polypeptide (DP 7-C): the Chengdayaki biological medicine science and technology development company is synthesized;
bFGF: purchased from Beijing Yiqiao Shenzhou technologies, Inc.;
the instrument comprises the following steps: malvern laser particle size detector ZEN 3600.
1. Effect of the Soybean lecithin/sodium deoxycholate/Hydrophobically modified polypeptide (DP7-C) ratio on the quality of Flexible liposomes
Selecting soybean lecithin: the ratio of deoxycholate sodium (mg/mg) is 70:50,70:40,70:30,70:20,70:10, and transfersomes are prepared respectively, and the clarity, particle size and protein activity are compared, and vitamin E accounting for 0.2% of lipid weight is added as antioxidant protectant.
Accurately weighing soybean lecithin, sodium deoxycholate and vitamin E according to the proportion, and dissolving in 4ml of chloroform/ethanol (1:1) solution; after mixing well, the organic solvent was evaporated off to form a lipid thin film in a 100ml round bottom flask, at 37 ℃ and 50 rpm under reduced pressure for 2 hours. Then 4ml of the aqueous solution was added to the flask together with bFGF protein at a final concentration of 0.1mg/ml, and the flask was rotated to hydrate the lipid film for 40min to prepare bFGF-containing flexible liposomes. Then, the solution is subjected to ultrasonic treatment for 3 minutes under the power of 85W by using a probe, and then is filtered by using a 0.2-micron filter membrane to obtain a flexible liposome solution, and the flexible liposome solution is stored at 4 ℃ for standby application in particle size and protein activity detection. The results are shown in Table 1.
TABLE 1 transfersome quality index test results
As a result: when the ratio of soybean lecithin: when the mass ratio of the sodium deoxycholate is 70:10, the clarity and the transparency of the solution are the worst, and the particle size is the largest. As the proportion of sodium deoxycholate increases, the clarity of the solution increases and the particle size decreases. However, when the ratio is higher than 70:30, bFGF activity is decreased.
The above experimental results show that the ratio (mg/mg) of soybean lecithin to sodium deoxycholate is 70: (20-40), the flexible liposome with better clarity, particle size and protein activity can be prepared. Wherein, when the ratio (mg/mg) of the soybean lecithin to the sodium deoxycholate is 70:30, the quality indexes of the flexible liposome in all aspects are optimal.
On the basis (the mass ratio of soybean lecithin to sodium deoxycholate is 70:30), hydrophobic modified polypeptides (DP7-C) with different proportions (proportion of the total lipid materials) are added in the hydration process: 0%, 1%, 2.5%, 5%, 7.5%, 10%, flexible liposomes of different formulations were prepared according to the procedure of example 1. The quality test results are shown in Table 2.
TABLE 2 Flexible Liposome quality index test results
| DP7-C(％) | Clarity of the product | Particle size (nm) | |
| 0 | Clarification | 95 | 70％ |
| 1 | Clarification | 110 | 75％ |
| 2.5 | Clarification | 106 | 72％ |
| 5 | |
121 | 80％ |
| 7.5 | Not clarifying it | 266 | 40％ |
| 10 | Not clarifying it | 302 | <20％ |
As a result: when the proportion of the hydrophobic modified polypeptide DP7-C is gradually increased (1% to 10%), the clarity of the flexible liposome is kept clear, the particle size is maintained at 100nm, and the protein activity is gradually increased. When the DP7-C ratio reaches 7.5%, the clarity of the solution decreases, the particle size increases, and the protein activity also decreases substantially.
The experimental results show that the proportion of the hydrophobic modified polypeptide DP7-C in the total lipid material is within 1-5%, and the flexible liposome with better clarity, particle size and protein activity can be prepared. Wherein, when the proportion of the hydrophobic modified polypeptide DP7-C in the total lipid material is 5%, the comprehensive quality evaluation of the flexible liposome is optimal.
In combination with the above experimental results, the ratio (mg/mg) of soybean lecithin to sodium deoxycholate is 70:30, and the ratio of hydrophobic modified polypeptide DP7-C modification is 5% which is the best formulation of the flexible liposome of the present invention.
2. Effect of the hydration Medium
bFGF has an isoelectric point of 10.0 or more and is strongly positively charged in a near-neutral solution, and therefore, a nonionic solvent is preferably used for hydration.
Transfersomes were prepared according to the procedure of example 1, using 5% glucose solution and distilled water as hydration media (both non-ionic solvents).
As a result: the distilled water group (clear, particle size <120nm) was significantly better than the 5% glucose group (more turbid, particle size >200nm) in both solution clarity and particle size.
The above experimental results show that the hydration medium is optimized with water, preferably distilled water.
Effect of bFGF content
The flexible liposomes were prepared by adding bFGF in the amounts shown in Table 3, respectively, and by the procedure of example 1. The quality test results are shown in Table 3.
TABLE 3 Flexible Liposome quality index test results
| bFGF content (. mu.g/ml) | Clarity of the product | Particle size (nm) | |
| 10 | |
90 | <20％ |
| 20 | Clarification | 98 | 40％ |
| 50 | Clarification | 115 | 70％ |
| 100 | Clarification | 135 | 80％ |
| 120 | Not clarifying it | 223 | 90％ |
As a result: as the bFGF content increases, the particle size of the solution also increases significantly. When the content is > 100. mu.g/ml, the particle size of the solution is >200nm, and in addition, the protein activity is improved accordingly.
The experimental results show that the carrier with better clarity, particle size and protein activity can be prepared when the final concentration of bFGF is within the range of 20-100 mug/ml. Wherein, when the final concentration of bFGF is 100 mug/ml (namely 0.1mg/ml), the comprehensive quality evaluation of the flexible liposome is best.
4. Effect of hydration time
Setting the hydration time to 10min, 30min, 45min, 60min and 90min respectively, and preparing the flexible liposome according to the operation method of the embodiment 1. The quality test results are shown in Table 4.
TABLE 4 Flexible Liposome quality index test results
| Hydration time | Clarity of the product | Particle size | Protein Activity |
| 10min | Clarification | 110nm | <20％ |
| 30min | Clarification | 102nm | 70％ |
| 45min | Clarification | 105nm | 70％ |
| 60min | Clarification | 120nm | 70％ |
| 90min | Clarification | 119nm | 70％ |
As a result: the hydration time has no great influence on the clarity and the particle size of the solution, but has influence on the protein activity, the protein activity is increased along with the prolonging of the hydration time, but the protein activity is not obviously increased when the hydration time is more than 30 min.
The experimental results show that the flexible liposome with better clarity, particle size and protein activity can be prepared when the hydration time is more than 30 min. The hydration time is 30min as the best from the feasibility point of the preparation process.
In summary, the parameters selected by the preparation process of the bFGF-containing flexible liposome are: the ratio of soybean lecithin to sodium deoxycholate is 7:3, the ratio of DP7-C is 5%, the final concentration of bFGF is 0.1mg/ml, the hydration medium is water, and the hydration time is 30 minutes. The specific preparation process comprises the following steps: accurately weighing soybean lecithin, sodium deoxycholate and vitamin E according to the formula, dissolving with chloroform/ethanol respectively, dissolving and mixing in a 250ml pear-shaped bottle, rotatably evaporating at room temperature for 2 hours, and placing the obtained film in a vacuum drying oven overnight. Distilled water and a proper amount of DP7-C are added in the next day, and the mixture is subjected to ultrasonic treatment for 30 minutes by a 400w probe to obtain an empty transfersome solution. Then adding bFGF slowly to the final concentration of 0.1mg/ml, incubating for 30 minutes at room temperature, filtering the obtained mother liquor for multiple times by using a 0.2 mu m polycarbonate membrane, adding an excipient of 5% mannitol, subpackaging in penicillin bottles, and freeze-drying to obtain the bFGF-containing flexible liposome 1-1 taking sodium deoxycholate as a surfactant, wherein the particle size of the bFGF-containing flexible liposome is about 89.37nm, as shown in Table 5, and the encapsulation rate of the product is 55.6% by an ultracentrifugation method.
Meanwhile, the surfactant is replaced by polysorbate 80 from sodium deoxycholate to prepare the DP7-C modified flexible liposome wrapping the bFGF, and the preparation process selects the parameters as follows: the ratio of soybean lecithin to polysorbate 80 is 7:3, the ratio of DP7-C is 5%, the final concentration of bFGF is 0.1mg/ml, the hydration medium is water, and the hydration time is 30 minutes. The specific preparation process comprises the following steps: accurately weighing soybean lecithin, polysorbate 80 and vitamin E according to the formula, dissolving with chloroform/ethanol (1:1), dissolving and mixing in 250ml pear-shaped bottle, rotary evaporating at room temperature for 2hr, and placing the obtained film in a vacuum drying oven overnight. Distilled water and a proper amount of DP7-C are added in the next day, and the mixture is subjected to ultrasonic treatment for 30 minutes by a 400w probe to obtain an empty transfersome solution. Then slowly adding bFGF to the final concentration of 0.1mg/ml, incubating for 30 minutes at room temperature, filtering the obtained mother liquor for multiple times by using a 0.2 mu m polycarbonate membrane, adding an excipient of 5% mannitol, subpackaging in penicillin bottles, and freeze-drying to obtain the bFGF-containing flexible liposome 1-2 taking polysorbate 80 as a surfactant, wherein the particle size of the bFGF-containing flexible liposome is about 86.29nm, the particle size diagram is shown in figure 1, and the encapsulation rate of the product is 51.23% by an ultracentrifugation method.
Experimental examples 1-2 preparation of PAL-DP7 modified Flexible liposomes containing bFGF
Experimental materials:
soybean lecithin: purchased from Lipoid GmbH;
sodium deoxycholate: purchased from biotechnology responsibility ltd of obozoxing, beijing;
polysorbate 80: merck, USA;
hydrophobically modified polypeptide PAL-DP 7: the Chengdayaki biological medicine science and technology development company is synthesized;
bFGF: purchased from Beijing Yiqiao Shenzhou technologies, Inc.;
the instrument comprises the following steps: malvern laser particle size detector ZEN 3600.
In this experimental example, the flexible liposome is modified by PAL-DP7, the surface active substances are respectively prepared by sodium deoxycholate and polysorbate 80, and the preparation process of the flexible liposome containing bFGF has the following parameters: the ratio of soybean lecithin to sodium deoxycholate or the ratio of soybean lecithin to polysorbate 80 is 7:3, the ratio of the hydrophobic modified polypeptide PAL-DP7 is 6%, the final concentration of bFGF is 0.1mg/ml, the hydration medium is water, and the hydration time is 30 minutes.
The specific preparation process comprises the following steps: accurately weighing soybean lecithin, sodium deoxycholate or polysorbate 80 vitamin E according to the formula, dissolving with chloroform/ethanol (1:1), dissolving and mixing in 250ml pear-shaped bottle, rotary evaporating at room temperature for 2hr, and placing the obtained film in a vacuum drying oven overnight. Distilled water and a proper amount of hydrophobic modified polypeptide PAL-DP7 are added in the next day, and a 400w probe is used for ultrasonic treatment for 30min to obtain a blank carrier solution. Then adding bFGF slowly to the final concentration of 0.1mg/ml, incubating for 30 minutes at room temperature, filtering the obtained mother liquor for multiple times by using a 0.2 mu m polycarbonate membrane, adding an excipient of 5% mannitol, subpackaging in penicillin bottles, and freeze-drying to obtain bFGF-containing flexible liposomes 1-3 using sodium deoxycholate as a surfactant and bFGF-containing flexible liposomes 1-4 using polysorbate 80 as a surfactant, wherein the particle diameters of the bFGF-containing flexible liposomes are about 73.5nm and 68.57nm respectively, as shown in Table 5, and the encapsulation efficiencies of the product are 54.9% and 56.17% respectively by ultracentrifugation. The flexible liposome modified by PAL-DP7 and using polysorbate 80 as surfactant has the smallest nanometer particle size and the best encapsulation efficiency, and the particle size diagram is shown in figure 2.
TABLE 5 Nanometric characterization of different compositions of Flexible liposomes containing bFGF
Experimental example 2 preparation of flexible liposome containing EGF
According to the research thought, the flexible liposome containing EGF is prepared at the same time.
Experimental example 2-1 preparation of DP7-C modified Flexible Liposome containing EGF
Experimental materials:
soybean lecithin: purchased from Lipoid GmbH;
sodium deoxycholate: purchased from biotechnology responsibility ltd of obozoxing, beijing;
polysorbate 80: merck, USA;
hydrophobic modified polypeptide DP 7-C: the Chengdayaki biological medicine science and technology development company is synthesized;
EGF: purchased from Beijing Yiqiao Shenzhou technologies, Inc.;
the instrument comprises the following steps: malvern laser particle size detector ZEN 3600.
In the experimental example, the flexible liposome is modified by DP7-C, the surface active substances are respectively prepared by sodium deoxycholate and polysorbate 80, and the preparation process of the flexible liposome containing EGF has the following parameters: the ratio of soybean lecithin to sodium deoxycholate or the ratio of soybean lecithin to polysorbate 80 is 7:3, the ratio of the hydrophobic modified polypeptide DP7-C is 5%, the final concentration of EGF is 0.05mg/ml, the hydration medium is water, and the hydration time is 30 minutes.
The specific preparation process comprises the following steps: accurately weighing soybean lecithin, sodium deoxycholate or polysorbate 80 vitamin E according to the formula, dissolving with chloroform/ethanol (1:2), dissolving and mixing in 250ml pear-shaped bottle, rotary evaporating at room temperature for 2hr, and placing the obtained film in a vacuum drying oven overnight. Adding distilled water and a proper amount of hydrophobic modified polypeptide DP7-C in the next day, and performing ultrasonic treatment for 30min by a 400w probe to obtain a blank carrier solution. And slowly adding EGF to a final concentration of 0.05mg/ml, incubating at room temperature for 30 minutes, filtering the obtained mother liquor for multiple times by using a 0.2 mu m polycarbonate membrane, adding an excipient of 5% mannitol, subpackaging in a penicillin bottle, and freeze-drying to obtain the flexible liposome 2-1 containing EGF taking sodium deoxycholate as a surfactant and the flexible liposome 2-2 containing EGF taking polysorbate 80 as a surfactant, wherein the particle diameters of the flexible liposome 2-1 and the flexible liposome 2-2 are respectively about 76.67nm and 75.13nm, and the encapsulation rates of the product are respectively 49.9% and 51.27% by an ultracentrifugation method as shown in Table 6. Wherein, the particle size diagram of the flexible liposome modified by DP7-C and taking polysorbate 80 as a surfactant is shown in figure 3.
Experimental example 2-2 preparation of PAL-DP7 modified Flexible liposomes containing EGF
Experimental materials:
soybean lecithin: purchased from Lipoid GmbH;
sodium deoxycholate: purchased from biotechnology responsibility ltd of obozoxing, beijing;
polysorbate 80: merck, USA;
hydrophobically modified polypeptide PAL-DP 7: the Chengdayaki biological medicine science and technology development company is synthesized;
EGF: purchased from Beijing Yiqiao Shenzhou technologies, Inc.;
the instrument comprises the following steps: malvern laser particle size detector ZEN 3600.
In this experimental example, the flexible liposome is modified by PAL-DP7, the surface active substances are respectively prepared by sodium deoxycholate and polysorbate 80, and the preparation process of the flexible liposome containing EGF has the following parameters: the ratio of soybean lecithin to sodium deoxycholate or the ratio of soybean lecithin to polysorbate 80 is 7:3, the ratio of the hydrophobic modified polypeptide PAL-DP7 is 6%, the final concentration of EGF is 0.0.5mg/ml, the hydration medium is water, and the hydration time is 30 minutes.
The specific preparation process comprises the following steps: accurately weighing soybean lecithin, sodium deoxycholate or polysorbate 80 vitamin E according to the formula, dissolving with chloroform/ethanol (1:2), dissolving and mixing in 250ml pear-shaped bottle, rotary evaporating at room temperature for 2hr, and placing the obtained film in a vacuum drying oven overnight. Distilled water and a proper amount of hydrophobic modified polypeptide PAL-DP7 are added in the next day, and a 400w probe is used for ultrasonic treatment for 30min to obtain a blank carrier solution. And slowly adding EGF to a final concentration of 0.05mg/ml, incubating at room temperature for 30 minutes, filtering the obtained mother liquor for multiple times by using a 0.2 mu m polycarbonate membrane, adding an excipient of 5% mannitol, subpackaging in a penicillin bottle, and freeze-drying to obtain flexible liposome 2-3 containing EGF taking sodium deoxycholate as a surfactant and flexible liposome 2-4 containing EGF taking polysorbate 80 as a surfactant, wherein the particle diameters of the flexible liposome 2-3 and the flexible liposome 2-4 are respectively 69.57nm and 64.18nm, and the encapsulation rates of the product are respectively 52.49% and 54.77% by using an ultracentrifugation method, as shown in Table 6. Wherein the flexible liposome modified by PAL-DP7 and with the smallest nanometer particle size and the best encapsulation efficiency is polysorbate 80 as surfactant, and the particle size diagram is shown in figure 4.
TABLE 6 Nanometric characterization of flexible liposomes of different compositions prepared with EGF
Experimental example 3 preparation of Flexible liposomes containing SOD
According to the research thought, the flexible liposome containing SOD is prepared at the same time.
Experimental example 3-1 preparation of DP7-C modified Flexible liposomes containing SOD
Experimental materials:
soybean lecithin: purchased from Lipoid GmbH;
sodium deoxycholate: purchased from biotechnology responsibility ltd of obozoxing, beijing;
polysorbate 80: merck, USA;
hydrophobic modified polypeptide DP 7-C: the Chengdayaki biological medicine science and technology development company is synthesized;
SOD (superoxide dismutase): purchased from Sigma, usa;
the instrument comprises the following steps: malvern laser particle size detector ZEN 3600.
In the experimental example, the flexible liposome is modified by DP7-C, the surface active substances are respectively prepared by sodium deoxycholate and polysorbate 80, and the parameters of the preparation process of the flexible liposome containing SOD are as follows: the ratio of soybean lecithin to sodium deoxycholate or the ratio of soybean lecithin to polysorbate 80 is 7:3, the ratio of the hydrophobic modified polypeptide DP7-C is 5%, the final concentration of SOD is 20000U/ml, the hydration medium is water, and the hydration time is 30 minutes.
The specific preparation process comprises the following steps: accurately weighing soybean lecithin, sodium deoxycholate or polysorbate 80 vitamin E according to the formula, dissolving with ethanol respectively, dissolving and mixing in a 250ml pear-shaped bottle, rotary evaporating at room temperature for 2 hours, and placing the obtained film in a vacuum drying oven overnight. Adding distilled water and a proper amount of hydrophobic modified polypeptide DP7-C in the next day, and performing ultrasonic treatment for 30min by a 400w probe to obtain a blank carrier solution. And slowly adding SOD to a final concentration of 20000U/ml, incubating at room temperature for 30min, filtering the obtained mother liquor with 0.2 μm polycarbonate membrane for multiple times, adding excipient 5% mannitol, subpackaging in penicillin bottles, and lyophilizing to obtain flexible liposome 3-1 containing SOD with sodium deoxycholate as surfactant and flexible liposome 3-2 containing SOD with polysorbate 80 as surfactant, wherein the particle diameters are about 71.38nm and 70.26nm, respectively, as shown in Table 7, and the encapsulation efficiencies of the product are 55.27% and 56.74% respectively by ultracentrifugation. Wherein, the particle size diagram of the flexible liposome modified by DP7-C and taking polysorbate 80 as a surfactant is shown in figure 5.
Experimental example 3-2 preparation of SOD-containing PAL-DP 7-modified Flexible liposomes
Experimental materials:
soybean lecithin: purchased from Lipoid GmbH;
sodium deoxycholate: purchased from biotechnology responsibility ltd of obozoxing, beijing;
polysorbate 80: merck, USA;
hydrophobically modified polypeptide PAL-DP 7: the Chengdayaki biological medicine science and technology development company is synthesized;
SOD (superoxide dismutase): purchased from Sigma, usa;
the instrument comprises the following steps: malvern laser particle size detector ZEN 3600.
In the experimental example, the flexible liposome is modified by PAL-DP7, the surface active substances are respectively prepared by sodium deoxycholate and polysorbate 80, and the parameters of the preparation process of the flexible liposome containing SOD are as follows: the ratio of soybean lecithin to sodium deoxycholate or the ratio of soybean lecithin to polysorbate 80 is 7:3, the ratio of the hydrophobic modified polypeptide PAL-DP7 is 6%, the final concentration of SOD is 20000U/ml, the hydration medium is water, and the hydration time is 30 minutes.
The specific preparation process comprises the following steps: accurately weighing soybean lecithin, sodium deoxycholate or polysorbate 80 vitamin E according to the formula, dissolving with ethanol respectively, dissolving and mixing in a 250ml pear-shaped bottle, rotary evaporating at room temperature for 2 hours, and placing the obtained film in a vacuum drying oven overnight. Distilled water and a proper amount of hydrophobic modified polypeptide PAL-DP7 are added in the next day, and a 400w probe is used for ultrasonic treatment for 30min to obtain a blank carrier solution. And then slowly adding SOD to a final concentration of 20000U/ml, incubating for 30 minutes at room temperature, filtering the obtained mother liquor for multiple times by using a 0.2-micron polycarbonate membrane, adding excipient 5% mannitol, subpackaging in penicillin bottles, and freeze-drying to obtain flexible liposome 3-3 containing SOD with sodium deoxycholate as a surfactant and flexible liposome 3-4 containing SOD with polysorbate 80 as a surfactant, wherein the particle diameters of the flexible liposome 3-3 and the flexible liposome 3-4 are respectively 69.18nm and 65.19nm, as shown in Table 7, and the encapsulation rates of the product are respectively 56.87% and 59.28% by using an ultracentrifugation method. Wherein the flexible liposome modified by PAL-DP7 and with the smallest nanometer particle size and the best encapsulation efficiency is polysorbate 80 as surfactant, and the particle size diagram is shown in figure 6.
TABLE 7 Nanometric characterization of the preparation of flexible liposomes of different compositions containing SOD
Example 2 in vitro transdermal effect verification of Flexible liposomes containing biomacromolecules of the present invention
In vitro transdermal experiment, an improved single-chamber Franz diffusion cell is adopted, mouse skin is used as the skin of the in vitro transdermal experiment, the transdermal absorption results of different flexible liposomes containing biological macromolecules are compared, samples are respectively taken at different time points, qualitative detection is respectively carried out by SDS-PAGE and Western Blot methods, or the concentration of the drugs in the transdermal receiving solution is respectively determined by ELISA method, and the accumulated transdermal quantity is calculated.
Test example 2-1 qualitative verification of transdermal Effect of bFGF-containing Flexible liposomes
The experimental example qualitatively determined the transdermal effect of DP7-C modified flexible liposomes containing bFGF using protein electrophoresis and Western Blot of the corresponding antibodies.
1. Material
Sample preparation: lyophilized powder of bFGF-containing flexible liposome prepared in test example 1 of example 1 (1-1);
the instrument comprises the following steps: a drug transdermal test diffusion instrument (Shanghai yellow sea drug test, model RYJ-6B);
materials: stripped mouse skin;
anti-bFGF antibody: purchased from Abcam.
2. Method of producing a composite material
Dissolving the freeze-dried powder of the bFGF-containing flexible liposome by using distilled water, and then adding the dissolved freeze-dried powder on a diffusion cell cover, wherein the different time periods are as follows: sampling from the diffusion cell for 0h, 1h, 2h, 4h, 8h and 24h, and storing at-20 ℃ to be tested.
Samples to be detected at each time point were separated by 12% SDS-PAGE, transferred to a membrane, and the presence of bFGF after permeation was detected by Western Blot using a specific anti-bFGF antibody.
3. Results
The SDS-PAGE results of the samples at different time periods are shown in FIG. 7A, and the Western Blot results are shown in FIG. 7B. Wherein Lane1 is a 0h sample, Lane2 is a 1h sample, Lane3 is a 2h sample, Lane4 is a 4h sample, Lane5 is an 8h sample, and Lane6 is a 24h sample.
The results showed that some bFGF protein penetrated the skin of mice at the beginning of 2 hours, and the proportion of bFGF protein penetrated the skin gradually increased with the lapse of time.
Test example 2-2 ELISA quantitative verification of transdermal Effect of bFGF-containing Flexible liposomes
In this test example, the transdermal effect of the bFGF-containing flexible liposome was quantitatively measured using an ELISA kit.
1. Material
Sample preparation: lyophilized powder of bFGF-containing liposomes prepared in test example 1 of example 1 (1-1, 1-2, 1-3, 1-4) and bFGF-containing liposomes that were not modified with DP7-C or PAL-DP7 were used as normal liposomes controls;
the instrument comprises the following steps: a drug transdermal test diffusion instrument (Shanghai yellow sea drug test, model RYJ-6B); an enzyme-labeling instrument: Bio-Rad, iMark;
materials: stripped mouse skin.
bFGF protein ELISA detection kit: purchased from R & D, usa.
2. Method of producing a composite material
2.1 the transdermal test of the sample is carried out according to the instructions of the diffusion instrument of the transdermal test, and a blank control with buffer solution added above is arranged at the same time, and the samples in the receiving pool are harvested respectively at 0hr, 1hr, 2hr, 4hr and 8hr of the test.
2.2ELISA detection: and (4) detecting samples at each time point according to the ELISA detection kit instruction, and calculating the bFGF content in the samples according to the standard curve.
3. Results
The ELISA detection kit using bFGF was used to quantitatively detect the bFGF content in the receiving pool samples at each time point, and the results are shown in FIG. 8. The results showed that the amount of bFGF penetrated the skin of mice increased gradually with time, and that DP7-C and PAL-DP7 modified flexible liposomes had better transdermal properties than the unmodified ordinary flexible liposomes.
Test examples 2-3 ELISA quantitative verification of transdermal Effect of EGF-containing Flexible liposomes
The test example quantitatively determined the transdermal effect of the flexible liposome containing EGF using an ELISA kit.
1. Material
Sample preparation: lyophilized powder of flexible liposomes of EGF (2-1, 2-2, 2-3, 2-4) prepared in Experimental example 2 of example 1 and flexible liposomes that were not modified with DP7-C or PAL-DP7 served as a common flexible liposome control;
the instrument comprises the following steps: a drug transdermal test diffusion instrument (Shanghai yellow sea drug test, model RYJ-6B); an enzyme-labeling instrument: Bio-Rad, iMark;
materials: stripped mouse skin.
EGF protein ELISA detection kit: purchased from R & D, usa.
2. Method of producing a composite material
2.1 the transdermal test of the sample is carried out according to the instructions of the diffusion instrument of the transdermal test, and a blank control with buffer solution added above is arranged at the same time, and the samples in the receiving pool are harvested respectively at 0hr, 1hr, 2hr, 4hr and 8hr of the test.
2.2ELISA assay: and (4) detecting samples at each time point according to the ELISA detection kit instruction, and calculating the EGF content in the samples according to the standard curve.
3. Results
The ELISA detection kit for EGF is adopted to quantitatively detect the content of EGF in the receiving pool sample at each time point, and the result is shown in figure 9. The results showed that the EGF amount passing through the skin of mice increased gradually with time, and that DP7-C and PAL-DP7 modified flexible liposomes had better transdermal properties than the unmodified common flexible liposomes.
Test examples 2-4 verification of transdermal Effect of SOD-containing Flexible liposomes
In this test example, the transdermal effect of the flexible liposome containing SOD was quantitatively measured by ELISA kit.
1. Material
Sample preparation: lyophilized powder of SOD flexible liposomes (3-1, 3-2, 3-3, 3-4) prepared in test example 3 of example 1 and flexible liposomes that were not modified with DP7-C or PAL-DP7 were used as a common flexible liposome control; (ii) a
The instrument comprises the following steps: a drug transdermal test diffusion instrument (Shanghai yellow sea drug test, model RYJ-6B); an enzyme-labeling instrument: Bio-Rad, iMark;
materials: stripped mouse skin.
SOD protein ELISA detection kit: purchased from Shanghai, West and Tang Biotech, Inc.
2. Method of producing a composite material
2.1 the transdermal test of the sample is carried out according to the instructions of the diffusion instrument of the transdermal test, and a blank control with buffer solution added above is arranged at the same time, and the samples in the receiving pool are harvested respectively at 0hr, 1hr, 2hr, 4hr and 8hr of the test.
2.2ELISA assay: and (4) detecting the samples at each time point according to the ELISA detection kit instruction, and calculating the SOD content in the samples according to the standard curve.
3. Results
The content of SOD in the receiving pool samples at each time point was quantitatively determined by using ELISA detection kit for SOD, and the results are shown in FIG. 10. The results showed that the amount of SOD penetrating the skin of mice increased gradually with the passage of time, and that DP7-C and PAL-DP7 modified flexible liposomes had better transdermal properties than the unmodified common flexible liposomes.
Example 3 in vivo transdermal effect verification of Flexible liposomes containing biomacromolecules of the present invention
This example uses immunohistochemistry to determine transdermal efficacy in vivo in the skin of mice containing DP7-C modified flexible liposomes of bFGF.
1. Material
Sample preparation: lyophilized powder of bFGF-containing flexible liposome (1-1) prepared in test example 1 of example 1;
animals: balb/c mice, 6-8 weeks old, female;
reagent: anti-bFGF antibodies were purchased from Abcam, Ready-to-use mouse SABC-AP immunohistochemistry kits.
2. Method of producing a composite material
The freeze-dried powder of the bFGF-containing flexible liposomes was applied to the skin of the dehaired mice in distilled water and sacrificed after 8 hours. Peeling off the skin coated with the sample, fixing, embedding in wax block, slicing, adopting an immunohistochemical method, and detecting the existence of bFGF in the tissue by using an anti-bFGF specific antibody and an immunohistochemical kit.
3. Results
The immunohistochemical assay results are shown in FIG. 11. The results of the skin sections after application showed that the sections applied with the flexible liposomes were stained yellow brown (positive) by specific anti-bFGF antibody (see fig. 11B), while the control skin was not stained (see fig. 11A), indicating that the flexible liposomes (1-1) were able to effectively penetrate the skin of mice with biomacromolecule bFGF after 8 hours.
Example 4 stability study of Flexible Liposome lyophilized powder containing biomacromolecule of the invention
Stability studies (including three aspects of accelerated tests and long-term tests) were performed on the bFGF-containing flexible liposome lyophilized powder (1-1) prepared in test example 1 of example 1. The samples were placed in a 37 ℃ drug stability test chamber for accelerated testing, and were placed for 1, 2, 3, and 6 months, respectively, and the properties of the lyophilized powder, including color, purity, and activity, were determined, with the results shown in table 8. And placing the sample in a drug stability test box at 25 ℃ for long-term test, respectively placing for 3, 6, 9, 12, 15, 18, 21 and 24 months, and respectively detecting the color, the purity and the activity of the freeze-dried powder, wherein the results are shown in Table 9.
TABLE 8 stability acceleration test of lyophilized powder (37 deg.C)
TABLE 9 stability Long-term test of lyophilized powder (25 deg.C)
The results show that the freeze-dried powder keeps the properties and the activity of the sample in the first 3 months of the accelerated test, the activity is reduced in the 6 th month, and the high-temperature storage is avoided as much as possible. The freeze-dried powder keeps better properties and activity in a long-term test at 25 ℃, and can ensure that the activity is not lost after 2 years of storage.
Example 5 preparation of essence containing Flexible liposomes of biological macromolecules according to the invention
The flexible liposome containing the biomacromolecule can be prepared into essence by using a solvent for use. The formula of the solvent is as follows: 0.3 percent of hyaluronic acid, 0.1 percent of allantoin, 0.5 percent of collagen, 0.1 percent of EDTA and 0.15 percent of multivitamin. The formula is aseptically subpackaged into 10-30 ml bottled solvents. In use, 1 bottle of the freeze-dried powder prepared in example 1 is dissolved in 1 bottle of the solvent to obtain the essence, and the essence is directly smeared on the face after the face is cleaned in the morning and evening.
The use effect of the essence containing the biomacromolecule flexible liposome is verified as follows:
subject: 1000 volunteers aged 30-60 years;
the experimental method comprises the following steps: volunteers were randomly divided into two groups, one group was the essence of the bFGF-containing flexible liposome (1-1) prepared in example 5, and the other group was the vehicle without flexible liposome. After cleaning the face in the morning and evening every day, the two groups of essences are respectively applied to the face and gently massaged until the essences are absorbed, and other skin care products are not applied. The trial period is 2-6 weeks, and a using effect statistical table is filled after trial, and the main indexes comprise functions of skin care, acne removal, speckle removal, sunburn repair, allergy resistance and the like.
The experimental results are as follows: the essence is tried for nearly 1000 people all over the country, such as Chengdu, Wenchang, Tibet, Chongqing, Wuhan, Hainan, Shenzhen and the like, the trial effect evaluation is better (see table 10 for details), and simultaneously 1 case of allergic symptoms does not appear, which indicates that the essence has good safety.
TABLE 10 results of using the essence of flexible liposome containing biomacromolecule of the present invention
From the above results, it can be seen that: the flexible liposome containing biomacromolecules can be applied to cosmetics, the transdermal efficiency is high, the effect is good, the prepared cosmetics show good effects in the aspects of wrinkle resistance, speckle removal, sunburn restoration and anti-allergy, and the application prospect is wide.
Sequence listing
<110> Shenzhen Shenshangke scientific and technology Limited
<120> flexible liposome cosmetic containing biomacromolecule and preparation method thereof
<130> A190059K
<141> 2019-01-30
<150> 201810150523.2
<151> 2018-02-13
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Val Gln Trp Arg Ile Arg Val Ala Val Ile Arg Lys
1 5 10
Claims (21)
1. A flexible liposome cosmetic comprising a biomacromolecule, characterized in that: is prepared by a polypeptide modified flexible liposome modified by hydrophobic modification and containing a biological macromolecular active ingredient; the hydrophobic modified polypeptide modified flexible liposome is prepared from the following raw materials in parts by weight: lecithin sodium deoxycholate or polysorbate 80-7: 2-4, 1-10 wt% of hydrophobic modified polypeptide, and biomacromolecule active ingredient; the hydrophobic modified polypeptide sequence is that NH is connected with the C end on the basis of SEQ ID NO 12And coupling cholesterol or palmitic acid to the nitrogen terminus of the polypeptide.
2. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: also contains antioxidant.
3. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 2, wherein: the antioxidant comprises at least one of vitamin C, vitamin E or coenzyme Q.
4. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 2, wherein: the weight percentage of the antioxidant is 0.1-1%.
5. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: the active ingredient of the biological macromolecule is growth factor or enzyme.
6. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: the biological macromolecule is at least one of fibroblast growth factor bFGF, keratinocyte growth factor KGF, vascular endothelial cell growth factor VEGF, superoxide dismutase SOD or lysozyme.
7. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: the weight percentage of the biological macromolecules is 1-10%.
8. The flexible liposome cosmetic containing biological macromolecules of claim 1, which is prepared from the following components in parts by weight: lecithin-sodium deoxycholate or polysorbate 80-7: 3, 0.5% of antioxidant and 2-10% of hydrophobization modified polypeptide.
9. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: the structure of the hydrophobic modified polypeptide is as follows:
wherein R is
10. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 6 wherein: the fibroblast growth factor bFGF accounts for 1-10% of the liposome by weight.
11. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 6 wherein: the fibroblast growth factor bFGF accounts for 2-8% of the weight of the liposome.
12. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 6 wherein: the fibroblast growth factor bFGF accounts for 5 percent of the weight of the liposome.
13. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 6 wherein: the superoxide dismutase SOD accounts for 1-5% of the liposome by weight.
14. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 6 wherein: the superoxide dismutase SOD accounts for 1-3% of the liposome by weight.
15. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 6 wherein: the superoxide dismutase SOD accounts for 2 percent of the weight of the liposome.
16. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: the flexible liposome cosmetic containing the biomacromolecule also comprises at least one of an emulsifier, a co-emulsifier, a skin conditioner, a whitening agent, a coloring agent, a humectant, a solubilizer, a surfactant, a preservative, a fragrance, an emollient, an anti-acne agent, a film forming agent, a thickener, a pH regulator, a buffering agent, a stabilizer or an ultraviolet absorbent which are relevant to the preparation of the cosmetic.
17. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: the flexible liposome cosmetic containing the biomacromolecule is in the dosage form of aqua, emulsion, paste, jelly, troche or aerosol.
18. A flexible liposome cosmetic containing biological macromolecules as claimed in claim 1, wherein: the flexible liposome cosmetic containing the biomacromolecule is in the dosage form of emulsion, essence, cream, facial mask, freeze-dried powder or facial cleanser.
19. A method of preparing a flexible liposome cosmetic comprising a biomacromolecule according to any of claims 1 to 18, comprising the steps of:
a. weighing lecithin, sodium deoxycholate or polysorbate 80 and vitamin E, placing into a reaction container, adding a solvent for dissolving, wherein the solvent is a mixture of chloroform and ethanol at a ratio of 1: 1-3 or is ethanol;
b. vacuum rotary evaporating to obtain liposome membrane, and drying;
c. adding hydrophobic modified polypeptide and distilled water into the liposome membrane, and performing ultrasonic hydration to obtain a blank liposome solution;
d. adding the bioactive macromolecules into the blank liposome solution, slightly shaking, and incubating at room temperature for 30-60 min to obtain the flexible liposome containing the bioactive macromolecules.
20. The method of preparing a flexible liposome cosmetic containing a biomacromolecule of claim 19, wherein: further comprising step e: extruding the incubated liposome to pass through a 0.1-0.45 mu m polycarbonate membrane for 4-8 times.
21. The method of preparing a flexible liposome cosmetic containing a biomacromolecule of claim 19, wherein: further comprising the step f: according to the dosage form requirement, the flexible liposome is further added with at least one of emulsifying agent, auxiliary emulsifying agent, skin conditioning agent, whitening agent, colorant, humectant, solubilizer, surfactant, antiseptic, aromatic, emollient, anti-acne agent, film forming agent, thickener, pH regulator, buffer, stabilizer or ultraviolet absorbent to prepare cosmetic dosage form.
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