CN111110699A

CN111110699A - Application of combination of polypeptide conjugate and epidermal stem cell exosome in medicines and cosmetics

Info

Publication number: CN111110699A
Application number: CN202010251262.0A
Authority: CN
Inventors: 彭菲; 李静
Original assignee: Beijing Yuehao Technology Development Co ltd
Current assignee: Guangzhou new era Cosmetics Co.,Ltd.
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2020-05-08
Anticipated expiration: 2040-04-01
Also published as: CN111110699B

Abstract

The invention relates to application of a combination of a polypeptide conjugate and an epidermal stem cell exosome in medicines and cosmetics.

Description

Application of combination of polypeptide conjugate and epidermal stem cell exosome in medicines and cosmetics

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of a combination of a polypeptide conjugate and an epidermal stem cell exosome in medicines and cosmetics.

Background

Exosomes (exosomes) are subcellular bilayer membrane vesicles with molecular diameters of 40-100 nm, which are formed by cells through a series of regulation processes of endocytosis, fusion and efflux and the like and can be secreted. The exosome is flat or spherical under a transmission electron microscope, the density of the exosome in a sucrose solution is 1.13-1.19 g/mL, and the density of the exosome is related to a cell source and is changed along with the protein content. The exosome does not contain DNA fragments, but contains proteins such as cytokines and growth factors similar to the source cells, and bioactive substances such as lipid, coding or non-coding RNA and the like, and has an important role in regulating and controlling the physiological functions of cells. The plasma membrane of exosomes is rich in cholesterol, sphingomyelin, ceramide, lipid rafts and phosphatidylserine; exosomes not only protect these bioactive substances from degradation and dilution in the extracellular environment, but also facilitate the long-range transport of these substances through interstitial fluid or blood, while specific surface ligands on the exosome membrane allow it to bind to receptor cells with high efficiency.

Research shows that exosomes have great relationship with skin-related diseases such as allergic diseases, melanoma, autoimmune diseases, wound healing and the like, and play important biological roles in various physiological and pathological processes. Melanoma recruits bone marrow-derived cells via exosomes, inducing the formation of a pre-metastatic niche. Can also enhance the permeability of the pulmonary vascular endothelium and promote the occurrence of pulmonary metastasis of mice. Studies have demonstrated that melanoma-derived exosomes rapidly enter mesenchymal stromal cells and promote their proliferation and migration. This effect may be beneficial for recruitment to more stromal cells in the tumor microenvironment, thus better supporting tumor growth and metastasis.

The study finds that the MSC-Exo can activate signal cascade channels such as protein kinase B (Akt) and extracellular regulatory protein kinase (ERK) and the like, and improves the expression quantity of factors such as Hepatocyte Growth Factor (HGF), insulin-like growth factor (IGF) -1 and Nerve Growth Factor (NGF) and the like to promote wound healing.

In addition, the experts recently point out that exosomes secreted by epidermal keratinocytes under ultraviolet radiation can regulate the expression of melanocyte-related genes through miRNA, enhance the activity of tyrosinase, promote melanogenesis and provide a new direction for the treatment of pigment diseases and the development of sunscreen products.

CN108451887A provides a composition for skin care, which comprises human umbilical cord mesenchymal stem cell-derived exosome, epidermal growth factor and sodium hyaluronate, and is used for moisturizing, repairing, anti-wrinkle and/or whitening human face or eyes by exerting synergistic effects of the three.

The applicant also disclosed in CN110680928A a cosmetic for skin whitening, which provides a polypeptide conjugate having a certain melanin inhibiting effect, but has a certain space for improvement, and has a great value in combination.

However, the skin cosmetic compositions for which the use of exosomes has been disclosed are not sufficiently studied on one hand, and on the other hand, there is still room for improvement in cosmetic effects.

Disclosure of Invention

The invention provides an exosome, and particularly provides an exosome of a mouse epidermal stem cell.

In another aspect of the invention, a preparation method of exosomes of mouse epidermal stem cells is provided, which mainly adopts trehalose with the final concentration of 0.5% and PEG 6000 with the final concentration of 8% to treat the exosomes, so that the extraction amount of the exosomes can be improved with high quality, and the exosomes have better extraction effect.

In another aspect, the present invention provides a method for preparing a specific exosome, comprising inoculating epidermal stem cells of a mouse to a 25mm flask in which NIH3T3 cells are grown, adding a special medium DMEM HAM F-12=3:1 mixed with 10% FBS, 20ng/ml EGF, 5ug/ml transferrin, 1.8 × 10^-4mo1/L adenine, 5ug/ml insulin and 0.4ug/ml hydrocortisone, pH = 7Changing the culture medium every 2 days at intervals of 0-7.4, and maintaining the culture environment at humidity>95%, temperature 37 ℃, CO₂The concentration was 5%. When the cells are 80% fused and can be passaged, NIH3T3 cells are removed by 0.02% EDTA, and then the cells are digested by 0.25% pancreatin and 0.02% EDTA for passage. Repeating the culture and amplification process for 3 times to complete the culture of a large number of epidermal stem cells.

In another aspect, the invention provides a method for preparing exosome, which comprises the steps of subpackaging the cultured cell supernatant into 50ml centrifuge tubes, centrifuging at normal temperature of 3000rpm for 20min, and removing cells and cell debris. The centrifuged supernatant was collected, passed through a 0.22 μm filtration membrane to remove large particles and vesicles, and added with trehalose at a final concentration of 0.5% and PEG 6000 at a final concentration of 8%, and allowed to stand overnight at 4 ℃. Centrifuging the PEG 6000 cell supernatant mixed solution standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the centrifugal tube, discarding the supernatant of the centrifugal tube, inversely buckling the centrifugal tube on absorbent paper, and air-drying for 5-10 min. According to the volume ratio of the precipitate to 0.1g to 10mL, adding a proper amount of PBS or double distilled water for resuspension, centrifuging again at 5000rpm for 20min, discarding the supernatant, and resuspending with 100ul of PBS to obtain an exosome solution.

In another aspect of the invention, a vesicle with nearly circular or elliptical membrane shape and uniform exosome size is provided, and the diameter of the vesicle is about 55-90 nm and mainly distributed in the range of 60-70 nm.

In addition, the invention provides an application of the exosome in inhibiting the synthesis of the melanin of the cell.

In another aspect, the present invention further provides a cosmetic composition comprising liposome-encapsulated exosomes and suitable cosmetic adjuvants.

Further, the liposome encapsulating the exosome may be contained in an amount of 0.001 to 10.0wt%, specifically 0.001 to 1.0wt%, more specifically 0.01 to 1.0wt%, even more specifically 0.01 to 0.1wt%, but is not limited thereto.

In one embodiment of the invention, 3% by weight lecithin is dispersed in an aqueous phase containing 0.01% by weight exosomes at room temperature (e.g. 15 ℃). Supercritical carbon dioxide is then used to form a reverse micelle emulsion (water/low temperature process carbon dioxide). Then, the reaction was terminated, and the supercritical carbon dioxide was evaporated under reduced pressure to remove the supercritical carbon dioxide phase, thereby obtaining a liposome suspension in which exosomes were encapsulated.

The present invention further provides a cosmetic using the exosome in combination with the compound represented by the formula (I) disclosed in the inventor's prior patent CN110680928A, and a method for producing the compound.

(formula I)

The cosmetic composition according to the above embodiment may comprise adjuvants commonly used in cosmetics or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, flavoring agents, surfactants, water, ionic or nonionic emulsifiers, fillers, chelating agents, preservatives, vitamins, retarding agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics. These adjuvants are incorporated in the amounts usually used in the cosmetic or dermatological field.

The cosmetic composition according to the above embodiment has an appearance comprising a cosmetically or dermatologically acceptable medium or base. The cosmetic composition may be in any form suitable for topical application. For example, cosmetic compositions may be provided in the form of solutions, gels, solids, pastes, anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules or ionic (liposomes) and nonionic vesicular dispersions, and these compositions may be prepared according to conventional methods in the art.

The cosmetic composition according to the above-described embodiment is preferably applied in a form absorbed into the skin using microneedles or the like, but is not limited thereto.

The present invention further provides a hydrogel containing exosomes. The hydrogel may be at least one hydrogel such as gelatin, alginate, chitosan, fibrin, elastin, hyaluronic acid, collagen, methylcellulose or collagen and methylcellulose hydrogels, but is not limited thereto.

In the conventional technique, a culture solution obtained during culturing of epidermal stem cells is used. However, the above embodiment is different in that the exosomes present in the culture liquid are separated and purified to be used as a cosmetic ingredient, without using the culture liquid itself.

Further, the cosmetic composition of exosomes from epidermal stem cells according to the above-described embodiments may be used as an agent for improving the appearance of scars. Since stem cell exosomes contain proteins and growth factors that induce cell proliferation and differentiation as well as skin regeneration, they can be applied to old wounds and acne scars to reduce scar formation or appearance. Therefore, when used as a preparation for improving scar tissue, it can be administered in the form of a spray, a gel-type ointment, and a patch containing exosomes or the like from stem cells.

Further, still another embodiment of the present invention provides a pharmaceutical composition comprising exosomes derived from stem cells as an active ingredient, and more specifically, a pharmaceutical composition for skin regeneration comprising exosomes derived from stem cells as an active ingredient. Accordingly, the cosmetic composition for skin regeneration according to one embodiment of the present invention, which comprises exosomes derived from stem cells as an active ingredient, may be used as a pharmaceutical composition.

Advantageous effects

The invention extracts and obtains the exosome from the epidermal stem cell, prepares the exosome into the corresponding cosmetic composition, has the effect of inhibiting the synthesis of melanin, and simultaneously proves that the cosmetic composition has the effects of removing freckles and preventing and treating acne through skin experiments.

Drawings

FIG. 1 size distribution diagram of epidermal stem cell exosomes.

FIG. 2 is a graph showing the concentration of melanin.

Detailed Description

The following further describes a specific embodiment of the present invention with reference to the drawings and technical solutions. The experimental materials not particularly emphasized in the following examples are all conventional experimental materials, and are not particularly required, and are all conventional materials readily available to those skilled in the art.

Example 1 culture of mouse epidermal Stem cells

Inoculating mouse epidermal stem cells (mouse epidermal stem cells, cat # BJ-ATCC0672, Shanghai Pongjing Co., Ltd.) to a 25mm flask in which NIH3T3 cells are grown, adding a special medium DMEM HAM F-12=3:1, and mixing with 10% FBS, 20ng/ml EGF, 5ug/ml transferrin, and 1.8X 10^-4mo1/L adenine, 5ug/ml insulin and 0.4ug/ml hydrocortisone, pH = 7.0-7.4, changing the solution every 2 days, maintaining the culture environment at humidity>95%, temperature 37 ℃, C0₂The concentration was 5%. When the cells are 80% fused and can be passaged, NIH3T3 cells are removed by 0.02% EDTA, and then the cells are digested by 0.25% pancreatin and 0.02% EDTA for passage. Repeating the culture and amplification process for 3 times to complete the culture of a large number of epidermal stem cells.

Example 2 extraction of epidermal stem cell exosomes

The cell supernatant cultured in example 1 was dispensed into 50ml centrifuge tubes and centrifuged at 3000rpm at room temperature for 20min to remove cells and cell debris. The centrifuged supernatant was collected, passed through a 0.22 μm filtration membrane to remove large particles and vesicles, and added with trehalose at a final concentration of 0.5% and PEG 6000 at a final concentration of 8%, and allowed to stand overnight at 4 ℃. Centrifuging the PEG 6000 cell supernatant mixed solution standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the centrifugal tube, discarding the supernatant of the centrifugal tube, inversely buckling the centrifugal tube on absorbent paper, and air-drying for 5-10 min. According to the volume ratio of the precipitate to 0.1g to 10mL, adding a proper amount of PBS or double distilled water for resuspension, centrifuging again at 5000rpm for 20min, discarding the supernatant, and resuspending with 100ul of PBS to obtain an exosome solution. The exosomes obtained by extraction are checked by a transmission electron microscope, and the result shows that the exosomes are shown to be in the form of small vesicles with nearly circular or elliptical membranes with uniform sizes under the transmission electron microscope, the diameters of the vesicles are about 55-90 nm, and the vesicles are mainly distributed in the range of 60-70nm as shown in figure 1. In addition, the total protein content of the extracted exosomes was determined using the BCA kit instructions. The final results showed that the amount of total protein in the exosomes extracted per 20ml of supernatant after epidermal stem cell culture was (1546. + -. 75). mu.g/L.times.10⁷cells。

Example 3 whitening Effect of combinations of exosomes derived from epidermal Stem cells and polypeptide Compounds

The whitening effect of the combination of exosomes and compounds was determined by the extent of inhibition of melanogenesis in mouse melanomas. Cultured melanoma cells (A875 (human melanoma cells), cat. No.: EY-X0770, Shanghai-Yigao Biotech Co., Ltd.) were cultured at 1X 10⁵Cells/well Density seeded in 96-well plates, 10mg/L of a compound of formula (I) added to exosomes containing final concentrations of 30 and 50ug/ml, respectively

(this compound was previously developed by the inventors, and is disclosed in formula (II) of CN110680928A, and its preparation method is example 1 of this patent) medium was cultured for 3 days in place of the medium, and a negative control was set in which no compound was added, and a blank control was set in which neither exosome nor compound was added, and only the medium was used for the treatment. After 3 days, the medium was recovered and centrifuged at 4500rpm for 10 minutes, and absorbance was measured at 405nm to calculate the amount of melanin released from the cells. The cells adhered to the plate were removed by applying trypsin (trypsin-EDTA), and the number of cells was measured, followed by centrifugation to recover the cells. The cells were washed once with PBS and centrifuged to obtain cell pellets. To the cell particles, 1ml of a 1N sodium hydroxide (NaOH) solution containing 10% dimethyl sulfoxide (DMSO) was added to dissolve melanin at 80 ℃ for 2 hours, and then the resultant was added to a 96-well plate, and absorbance was measured at 405 nm. Melanin was quantified using the measured absorbance and normalized to the protein concentration of the sample to determine the concentration of synthetic melanin. Exosomes were used at concentrations of 30 and 50ug/ml on melanoma cells and the extent of melanin synthesis was examined. As a result, FIG. 2 demonstrates that melanin synthesis is inhibited in the presence of exosomes at all concentrations, the extracellular melanin concentration is only less than 10ug/mg at maximum, and, in the presence of the compound of formula (I), exosomes are specificHas synergistic and better melanin inhibiting effect, the concentration of the melanin inhibiting agent is less than 2 ug/mg, and the melanin inhibiting agent is obviously inhibited compared with a control.

Example 4 preparation of cosmetic and skin test thereof

3% by weight of lecithin was dispersed in an aqueous phase containing 0.01% by weight of the aqueous phase prepared in example 2 at room temperature (15 ℃), and then a reverse micelle emulsion was prepared using supercritical carbon dioxide. Subsequently, the reaction is terminated, and the supercritical carbon dioxide is evaporated under reduced pressure to remove the supercritical carbon dioxide phase, and a liposome suspension at 4 ℃ or lower in which exosomes from stem cells are encapsulated is obtained. The suspension was combined with the components of table 1 to prepare the corresponding cream.

TABLE 1 amount of each component of the cream

Composition comprising a metal oxide and a metal oxide	(wt%)
		Stearic acid	4
Cetyl alcohol	1
		Lanolin alcohol	2
Liquid paraffin	8
		Cyclomethicone	5
Polyoxyethylene monooleate	3
		Hexanediol	2
Glycerol	4
		Triethylamine	5
Carbomer	0.2
		The aforementioned liposome suspension	0.01
A compound of formula (I)	0.01
		Purified water	Excess material

(1) Selecting 30 acne volunteer patients, dividing into three groups, cleaning face before treatment, wiping with hot towel, treating group A with adapalene gel, applying adapalene gel to affected part every night, and continuously using for 4 weeks; group B was treated with the cosmetic composition of Table 1 by applying the cosmetic composition to the affected part every night for 4 weeks; group C was treated with the blank cosmetic composition of Table 1 without liposome suspension, applied to the affected area the same night for 4 weeks; the treatment effect is judged according to the effective rate before and after the treatment of the skin acne, and observation is continuously carried out for 4 weeks.

The change of the degree of skin damage before and after treatment was used as a criterion for determining the effect. The reduction rate of the front and back skin lesions is (number of rash before treatment-number of rash after treatment)/number of rash before treatment x 100%. Healing, wherein the skin damage reduction rate is more than or equal to 90 percent; has obvious effect, the skin damage reduction rate is 70 to 89 percent; improvement, the skin damage reduction rate is 30 to 69 percent; the skin damage reduction rate is not more than 30 percent. The observation result shows that the effective rates of A, B, C three groups are respectively 60.74%, 94.87% and 83.88%, and meanwhile, the patients do not have allergy or other adverse reactions. It was shown that the cosmetic compositions of Table 1 were effective in treating acne and better than adapalene gel.

(2) Selecting 30 cases of freckle volunteer patients, dividing into three groups, cleaning face before treatment, wiping with hot towel, treating group A with three-dimensional peptide, applying three-dimensional peptide on affected part every night, and continuously using for 3 months; group B was treated with the cosmetic composition of Table 1 by applying the cosmetic composition of Table 1 to the affected part every night for 3 months; group C treatment with the blank cosmetic composition of Table 1 without liposome suspension applied to the affected area every night for 3 months; the affected part was observed for change in pigmentation after 3 months. The results show that the effective rates of A, B, C three groups are respectively 30.55%, 99.86% and 85.21%, and the patients have no allergy or other adverse reactions. The results show that the cosmetic composition of table 1 has a remarkable effect of treating freckles.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

Claims

1. The application of the composition of the polypeptide conjugate and the epidermal stem cell exosome in preparing medicines or cosmetics for inhibiting the synthesis of cell melanin is characterized in that the structural formula of the polypeptide conjugate is shown as the formula (I):

formula I:

。

2. the use of claim 1, wherein: the preparation method of the epidermal stem cell exosome comprises the following steps: the supernatant of the cultured epidermal stem cells is respectively filled into 50ml centrifuge tubes, and is centrifuged for 20min at the normal temperature of 3000rpm to remove the cells and cell debris; collecting the centrifuged supernatant, filtering with 0.22 μm filter membrane to remove large particles and vesicles, adding trehalose with a final concentration of 0.5% and PEG 6000 with a final concentration of 8%, and standing at 4 deg.C overnight; centrifuging the PEG 6000 cell supernatant mixed solution standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the centrifugal tube, discarding the supernatant of the centrifugal tube, inversely buckling the centrifugal tube on absorbent paper, and air-drying for 5-10 min; according to the volume ratio of the precipitate to 0.1g to 10mL, adding a proper amount of PBS or double distilled water for resuspension, centrifuging again at 5000rpm for 20min, discarding the supernatant, and resuspending with 100ul of PBS to obtain an exosome solution.

3. Use according to claim 2, characterized in that: the exosome presents a small vesicle with a nearly circular or elliptical membrane shape and uniform size, and the diameter is about 55-90 nm.

4. A facial cream characterized by: the preparation method comprises the following steps: preparing liposome suspension containing exosome, polypeptide conjugate shown in formula (I) and appropriate cream auxiliary materials into corresponding cream;

wherein the structural formula of the polypeptide conjugate of the formula (I) is

，

The preparation method of the liposome suspension comprises the following steps: obtaining a liposome suspension in which exosomes are encapsulated by dispersing 3% by weight of lecithin in an aqueous phase containing 0.01% by weight of exosomes, then forming a reverse micelle emulsion using supercritical carbon dioxide, then terminating the reaction, and evaporating the supercritical carbon dioxide under reduced pressure to remove the supercritical carbon dioxide phase;

the preparation method of the exosome comprises the following steps: the supernatant of the cultured epidermal stem cells is respectively filled into 50ml centrifuge tubes, and is centrifuged for 20min at the normal temperature of 3000rpm to remove the cells and cell debris; collecting the centrifuged supernatant, filtering with 0.22 μm filter membrane to remove large particles and vesicles, adding trehalose with a final concentration of 0.5% and PEG 6000 with a final concentration of 8%, and standing at 4 deg.C overnight; centrifuging the PEG 6000 cell supernatant mixed solution standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the centrifugal tube, discarding the supernatant of the centrifugal tube, inversely buckling the centrifugal tube on absorbent paper, and air-drying for 5-10 min; according to the volume ratio of the precipitate to 0.1g to 10mL, adding a proper amount of PBS or double distilled water for resuspension, centrifuging again at 5000rpm for 20min, discarding the supernatant, and resuspending with 100ul of PBS to obtain an exosome solution.

5. The cream of claim 4, wherein: the exosome presents a small vesicle with a nearly circular or elliptical membrane shape and uniform size, and the diameter is about 55-90 nm.

6. The cream of any one of claims 4-5, wherein: the face cream also contains adjuvants commonly used in dermatology: fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, flavoring agents, surfactants, water, ionic or nonionic emulsifiers, fillers, chelating agents, preservatives, vitamins, retarding agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics.

7. A facial cream characterized by: the weight percentages of the components are as follows:

4% of stearic acid, 1% of cetyl alcohol, 2% of lanolin alcohol, 8% of liquid paraffin, 5% of cyclomethicone, 3% of polyoxyethylene monooleate, 2% of hexanediol, 4% of glycerol, 5% of triethylamine, 0.2% of carbomer, 0.01% of liposome suspension, 0.01% of polypeptide conjugate shown in the formula (I) and the balance of purified water,

the preparation method of the exosome comprises the following steps: the supernatant of the cultured epidermal stem cells is respectively filled into 50ml centrifuge tubes, and is centrifuged for 20min at the normal temperature of 3000rpm to remove the cells and cell debris; collecting the centrifuged supernatant, filtering with 0.22 μm filter membrane to remove large particles and vesicles, adding trehalose with a final concentration of 0.5% and PEG 6000 with a final concentration of 8%, and standing at 4 deg.C overnight; centrifuging the PEG 6000 cell supernatant mixed solution standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the centrifugal tube, discarding the supernatant of the centrifugal tube, inversely buckling the centrifugal tube on absorbent paper, and air-drying for 5-10 min; adding a proper amount of PBS or double distilled water according to the volume ratio of 0.1g to 10mL according to the amount of the precipitate for heavy suspension, centrifuging for 20min at 5000rpm again, discarding the supernatant, and carrying out heavy suspension by using 100ul of PBS to obtain an exosome solution;

the structural formula of the polypeptide conjugate of the formula (I) is

。