CN111249211A

CN111249211A - Freeze-drying patch containing stem cell exosomes and preparation method and application thereof

Info

Publication number: CN111249211A
Application number: CN202010056135.5A
Authority: CN
Inventors: 戴博; 田丽军; 刘莹; 何婷婷; 李敏; 刘炜琳; 聂苏秦
Original assignee: Xi'an Jiuzhou Regenerative Medicine Group Co Ltd
Current assignee: Xi'an Jiuzhou Regenerative Medicine Group Co Ltd
Priority date: 2020-01-18
Filing date: 2020-01-18
Publication date: 2020-06-09

Abstract

The invention discloses a freeze-drying patch containing stem cell exosomes and a preparation method and application thereof, and belongs to the fields of cell bioengineering technology and medical science and beauty. The freeze-drying patch containing the stem cell exosomes comprises the following components: umbilical cord mesenchymal stem cell exosome, sodium alginate, trehalose, hydrolyzed sodium hyaluronate and deionized water. The preparation method of the freeze-dried patch comprises the following steps: soaking and swelling sodium alginate, trehalose and hydrolyzed sodium hyaluronate with part of deionized water, and stirring and mixing with the rest deionized water and the umbilical cord mesenchymal stem cell exosomes to obtain a mixed solution; and filling the mixed solution into a mold, placing the mold filled with the mixed solution into a vacuum freeze dryer for freeze-drying treatment, and then demolding to obtain the freeze-dried patch. The freeze-drying patch prepared by adding the umbilical cord mesenchymal stem cell exosome has high active matter content and convenient use, and has the effects of reducing wrinkles, improving skin glossiness and improving skin softness.

Description

Freeze-drying patch containing stem cell exosomes and preparation method and application thereof

Technical Field

The invention relates to the fields of cell bioengineering technology and medical science and beauty, in particular to a freeze-drying patch containing a stem cell exosome, and a preparation method and application thereof.

Background

The exosome is a nano-scale biological vesicle released into an extracellular environment after fusion of a multivesicular outer membrane and a plasma membrane, has the diameter of about 30-150 nm, can transport protein, lipid, nucleic acid and the like, participates in the processes of intercellular communication, cell migration, immune regulation and the like, and plays an important role in regulating the physiological and pathological processes of an organism.

The human umbilical cord mesenchymal stem cell is a stem cell separated from fetal umbilical cord Wharton's jelly and plays an important role in regenerative medicine. Compared with stem cells from other sources, the human umbilical cord mesenchymal stem cells have the advantages of non-invasive collection, low immunogenicity, easy in-vitro amplification, few ethical problems and the like, and have wide application prospects in the field of cell therapy.

The human umbilical cord mesenchymal stem cell exosome is a hot point of research in recent years, and carries partial information factors (mainly regeneration factors and various bioactive factors) of human umbilical cord mesenchymal stem cells, and can mediate cell-cell substance transfer and information conduction, so the human umbilical cord mesenchymal stem cell exosome has the receptor cell reparative effects of promoting fibroblast proliferation and migration, promoting synthesis of type I collagen and elastin, promoting reconstruction of extracellular matrix, inhibiting apoptosis, cell peroxidation and the like, and has the effects of resisting aging, preventing skin from being dark and inhibiting wrinkle generation.

At present, stem cell exosome products (namely cell-related factor products and the like) basically exist in a freeze-dried powder form, and the stem cell exosome freeze-dried powder needs to be added with a large amount of excipients such as mannitol and the like, so that cells are easily dehydrated, and the problems of low active substance content, poor effect and the like exist.

Disclosure of Invention

An object of an embodiment of the present invention is to provide a lyophilized patch containing a stem cell exosome, so as to solve the problems mentioned in the background art.

In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:

a freeze-drying patch containing stem cell exosomes comprises the following components in parts by weight: 0.0005-0.005 part of umbilical cord mesenchymal stem cell exosome, 0.1-1.5 parts of sodium alginate, 0.05-1 part of trehalose, 0.05-0.2 part of hydrolyzed sodium hyaluronate and 97.295-99.7995 parts of deionized water.

As a preferred scheme of the embodiment of the invention, the freeze-drying patch comprises the following components in parts by weight: 0.001-0.003 part of umbilical cord mesenchymal stem cell exosome, 0.8-1.2 parts of sodium alginate, 0.4-0.6 part of trehalose, 0.08-0.12 part of hydrolyzed sodium hyaluronate and 98.077-98.719 parts of deionized water.

As another preferable scheme of the embodiment of the invention, the preparation method of the umbilical cord mesenchymal stem cell exosome comprises the following steps:

culturing the P2 generation umbilical cord mesenchymal stem cells to obtain supernatant;

carrying out centrifugal extraction on the culture supernatant to obtain a precipitate;

and washing the precipitate, and then carrying out heavy suspension by using normal saline to obtain the umbilical cord mesenchymal stem cell exosome.

Another object of the embodiments of the present invention is to provide a method for preparing the lyophilized patch containing the stem cell exosomes, which includes the following steps:

weighing umbilical cord mesenchymal stem cell exosomes, sodium alginate, trehalose, hydrolyzed sodium hyaluronate and deionized water according to the weight parts for later use;

soaking and swelling the sodium alginate, the trehalose and the hydrolyzed sodium hyaluronate with part of deionized water, and then stirring and mixing the soaked and swollen sodium alginate, the trehalose and the hydrolyzed sodium hyaluronate with the rest of deionized water and the umbilical cord mesenchymal stem cell exosomes to obtain a mixed solution;

and filling the mixed solution into a mold, placing the mold filled with the mixed solution into a vacuum freeze dryer for freeze-drying treatment, and then demolding to obtain the freeze-dried patch.

As another preferable aspect of the embodiment of the present invention, the step of placing the mold filled with the mixed solution in a vacuum freeze dryer to perform freeze drying includes:

placing the mould filled with the mixed solution at the temperature of minus 20 to minus 10 ℃ for primary pre-freezing treatment, and then placing the mould at the temperature of minus 50 to minus 30 ℃ for secondary pre-freezing treatment;

and (3) placing the mold subjected to the secondary pre-freezing treatment at a temperature of-5 ℃ for primary drying treatment, and then placing the mold at a temperature of 10-30 ℃ for secondary drying treatment.

As another preferable scheme of the embodiment of the present invention, the mold includes a plate bottom, a mold hole and a plate cover, the plate bottom and the plate cover are connected in an inosculating manner, and the mold hole is located on the plate bottom and has a cylindrical structure; the diameter of the die hole is 5-50 mm, and the height of the die hole is 1-10 mm; and 1-100 die holes are formed in the bottom of the plate.

As another preferable scheme of the embodiment of the invention, in the step of placing the mould filled with the mixed solution at the temperature of-20 to-10 ℃ for primary pre-freezing treatment and then placing the mould at the temperature of-50 to-30 ℃ for secondary pre-freezing treatment, the duration of the primary pre-freezing treatment is 20 to 50min, and the duration of the secondary pre-freezing treatment is 50 to 80 min.

In another preferable embodiment of the invention, in the step of performing the primary drying treatment by placing the mold after the secondary pre-freezing treatment at a temperature of-5 to 5 ℃ and then performing the secondary drying treatment at a temperature of 10 to 30 ℃, the duration of the primary drying treatment is 400 to 700min, and the duration of the secondary drying treatment is 200 to 500 min.

In another preferable embodiment of the invention, in the step of performing the primary drying treatment at a temperature of-5 to 5 ℃ and then performing the secondary drying treatment at a temperature of 10 to 30 ℃, the vacuum degree of the primary drying treatment is 0.2 to 0.3mbar, and the vacuum degree of the secondary drying treatment is less than 0.01 mbar.

Another object of the embodiments of the present invention is to provide a freeze-dried patch prepared by the above preparation method.

As another preferable scheme of the embodiment of the invention, the freeze-dried paste is a cylindrical tablet, the diameter is 5-50 mm, and the height is 0.5-5 mm.

Another object of the embodiments of the present invention is to provide an application of the above-mentioned lyophilized patch in preparing a skin-caring product.

Compared with the prior art, the embodiment of the invention has the beneficial effects that:

the embodiment of the invention provides a freeze-drying patch containing human umbilical cord mesenchymal stem cell exosomes, which has the effects of fading wrinkles, improving skin glossiness and improving skin softness. In addition, the embodiment of the invention prepares the umbilical cord mesenchymal stem cell exosome into the freeze-dried patch which is convenient to use and store, has few auxiliary materials, does not need to add excipients such as mannitol and the like, and has the characteristics of high active substance content, good effect and the like.

Drawings

FIG. 1 is a graph showing the results of sodium fluorescein test in test animals before and after a stimulus test.

Fig. 2 is a comparative plot of skin condition of volunteers example 1 and example 2 before and after application of the lyophilized patch provided in example 6 of the present invention.

Fig. 3 is a comparative graph of skin condition of volunteers examples 3-5 before and after application of the lyophilized patch provided in example 6 of the present invention.

Fig. 4 is a safety and efficacy evaluation chart of the freeze-dried patch provided in embodiment 6 of the present invention.

Fig. 5 is a graph showing evaluation of the improvement in skin softness of the freeze-dried patch according to example 6 of the present invention.

Fig. 6 is a graph showing the evaluation of the freeze-dried patch according to example 6 of the present invention on the improvement of skin glossiness.

Fig. 7 is a graph showing the evaluation of improvement in skin wrinkles by the freeze-dried patch according to example 6 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment provides a preparation method of umbilical cord mesenchymal stem cell exosomes, which comprises the following steps:

(1) collecting umbilical cord tissues of a full-term healthy infant, and storing the umbilical cord tissues in a sterile bottle filled with phosphate buffer saline solution (containing 100U/mL streptomycin) for later use (requiring 4h for treatment); then, the vein and artery of the umbilical cord tissue were peeled off, and Wharton's jelly was peeled off and cut into 2mm pieces³The cells are placed in a culture bottle to be uniformly spread, a small amount of complete culture medium is added to keep moist, the existing DMEM/F12 culture medium (containing 15% fetal calf serum) can be adopted as the culture medium, enough culture medium is supplemented after 24 hours, then the culture medium is changed every 3 days, the cells grow to about 90% fusion degree for passage, and the culture medium is cultured by using D MEM/F12 culture medium (containing 10% fetal calf serum) after passage; and after the 3 rd-5 th generation cells grow to about 80% density, replacing the cells with serum-free DMEM/F12 culture medium, continuing culturing for 24 hours, and collecting culture supernatant to obtain the culture solution.

(2) Centrifuging the culture solution at 4 deg.C under 300g centrifugal force for 10min, and retaining supernatant A; centrifuging the supernatant A at 4 deg.C under 2000g centrifugal force for 10min, and retaining the supernatant B; centrifuging the supernatant B at 4 deg.C under 10000g centrifugation force for 30min, and retaining the supernatant C; centrifuging the supernatant C at 4 deg.C under 100000g for 70min, discarding the supernatant, and collecting the precipitate.

(3) And (3) centrifuging and washing the collected precipitate once by using phosphate buffered saline solution, and then carrying out heavy suspension by using normal saline to obtain the umbilical cord mesenchymal stem cell exosome, wherein the umbilical cord mesenchymal stem cell exosome can be stored in a refrigerator at the temperature of-80 ℃ for later use.

Example 2

The embodiment provides a freeze-drying patch containing stem cell exosomes and a preparation method thereof, and the preparation method of the freeze-drying patch comprises the following steps:

(1) weighing 0.0005g of umbilical cord mesenchymal stem cell exosome, 0.1g of sodium alginate, 0.05g of trehalose, 0.05g of hydrolyzed sodium hyaluronate and 99.7995g of deionized water for later use.

(2) Soaking and swelling the sodium alginate, the trehalose and the hydrolyzed sodium hyaluronate with part of deionized water, and then stirring and mixing with the rest deionized water and the umbilical cord mesenchymal stem cell exosomes to obtain a mixed solution.

(3) And (3) filling the mixed solution into a die hole of a die by using a liquid transfer gun, freeze-drying the die filled with the mixed solution in a freeze dryer, and then demolding to obtain the freeze-dried patch. The die comprises a plate bottom, a die hole and a plate cover, wherein the plate bottom and the plate cover are connected in an inosculating manner, and the die hole is positioned on the plate bottom and is of a cylindrical structure; the diameter of the die hole is 5mm, and the height of the die hole is 1 mm; the bottom of the plate is provided with 100 die holes, and the prepared freeze-drying paste is a cylindrical tablet with the diameter of 5mm and the height of 0.5 mm. In addition, the steps of the lyophilization process are as follows: placing the mould filled with the mixed solution at the temperature of minus 10 ℃ for primary pre-freezing treatment, and then placing the mould at the temperature of minus 30 ℃ for secondary pre-freezing treatment; placing the mold after the secondary pre-freezing treatment at the temperature of 0 ℃ for primary drying treatment, and then placing the mold at the temperature of 10 ℃ for secondary drying treatment, wherein the specific process parameters are set as the following table 1:

TABLE 1

Phases	Temperature/. degree.C	Time/min	Duration/min	Vacuum degree/mbar
					One-time pre-freezing	-10	40	20	-
Secondary pre-freezing	-30	40	50	-
					Primary drying	0	40	400	0.25
Secondary drying	10	40	200	0.009

Example 3

(1) weighing 0.005g of umbilical cord mesenchymal stem cell exosome, 1.5g of sodium alginate, 1g of trehalose, 0.2g of hydrolyzed sodium hyaluronate and 97.295g of deionized water for later use.

(3) And (3) filling the mixed solution into a die hole of a die by using a liquid transfer gun, freeze-drying the die filled with the mixed solution in a freeze dryer, and then demolding to obtain the freeze-dried patch. The die comprises a plate bottom, a die hole and a plate cover, wherein the plate bottom and the plate cover are connected in an inosculating manner, and the die hole is positioned on the plate bottom and is of a cylindrical structure; the diameter of the die hole is 50mm, and the height of the die hole is 10 mm; the bottom of the plate is provided with 1 die hole, and the prepared freeze-drying paste is a cylindrical tablet with the diameter of 50mm and the height of 5 mm. In addition, the steps of the lyophilization process are as follows: placing the mould filled with the mixed solution at the temperature of minus 20 ℃ for primary pre-freezing treatment, and then placing the mould at the temperature of minus 50 ℃ for secondary pre-freezing treatment; placing the mold after the secondary pre-freezing treatment at the temperature of 0 ℃ for primary drying treatment, and then placing the mold at the temperature of 30 ℃ for secondary drying treatment, wherein the specific process parameters are set as the following table 2:

TABLE 2

Phases	Temperature/. degree.C	Time/min	Duration/min	Vacuum degree/mbar
					One-time pre-freezing	-20	70	50	-
Secondary pre-freezing	-50	70	80	-
					Primary drying	0	80	700	0.25
Secondary drying	30	80	500	0.007

Example 4

(1) weighing 0.001g of umbilical cord mesenchymal stem cell exosome, 0.8g of sodium alginate, 0.4g of trehalose, 0.08g of hydrolyzed sodium hyaluronate and 98.719g of deionized water for later use.

(3) And (3) filling the mixed solution into a die hole of a die by using a liquid transfer gun, freeze-drying the die filled with the mixed solution in a freeze dryer, and then demolding to obtain the freeze-dried patch. The die comprises a plate bottom, a die hole and a plate cover, wherein the plate bottom and the plate cover are connected in an inosculating manner, and the die hole is positioned on the plate bottom and is of a cylindrical structure; the diameter of the die hole is 50mm, and the height of the die hole is 10 mm; the bottom of the plate is provided with 1 die hole, and the prepared freeze-drying paste is a cylindrical tablet with the diameter of 50mm and the height of 5 mm. In addition, the steps of the lyophilization process are as follows: placing the mould filled with the mixed solution at the temperature of minus 20 ℃ for primary pre-freezing treatment, and then placing the mould at the temperature of minus 50 ℃ for secondary pre-freezing treatment; placing the mold after the secondary pre-freezing treatment at a temperature of-5 ℃ for primary drying treatment, and then placing the mold at a temperature of 10 ℃ for secondary drying treatment, wherein the specific process parameters are set as the following table 3:

TABLE 3

Phases	Temperature/. degree.C	Time/min	Duration/min	Vacuum degree/mbar
					One-time pre-freezing	-20	60	40	-
Secondary pre-freezing	-50	60	70	-
					Primary drying	-5	70	60	0.2
Secondary drying	10	70	400	0.005

Example 5

(1) weighing 0.003g of umbilical cord mesenchymal stem cell exosome, 1.2g of sodium alginate, 0.6g of trehalose, 0.12g of hydrolyzed sodium hyaluronate and 98.077g of deionized water for later use.

(3) And (3) filling the mixed solution into a die hole of a die by using a liquid transfer gun, freeze-drying the die filled with the mixed solution in a freeze dryer, and then demolding to obtain the freeze-dried patch. The die comprises a plate bottom, a die hole and a plate cover, wherein the plate bottom and the plate cover are connected in an inosculating manner, and the die hole is positioned on the plate bottom and is of a cylindrical structure; the diameter of the die hole is 50mm, and the height of the die hole is 10 mm; the bottom of the plate is provided with 1 die hole, and the prepared freeze-drying paste is a cylindrical tablet with the diameter of 50mm and the height of 5 mm. In addition, the steps of the lyophilization process are as follows: placing the mould filled with the mixed solution at the temperature of minus 10 ℃ for primary pre-freezing treatment, and then placing the mould at the temperature of minus 30 ℃ for secondary pre-freezing treatment; and (3) placing the mould subjected to the secondary pre-freezing treatment at the temperature of 5 ℃ for primary drying treatment, and then placing the mould at the temperature of 30 ℃ for secondary drying treatment, wherein the specific process parameters are set as shown in the following table 4:

TABLE 4

Phases	Temperature/. degree.C	Time/min	Duration/min	Vacuum degree/mbar
					One-time pre-freezing	-10	60	30	-
Secondary pre-freezing	-30	60	60	-
					Primary drying	5	60	500	0.3
Secondary drying	30	60	300	0.005

Example 6

(1) weighing 0.002g of umbilical cord mesenchymal stem cell exosome, 1g of sodium alginate, 0.5g of trehalose, 0.1g of hydrolyzed sodium hyaluronate and 98.698g of deionized water for later use.

(3) And (3) filling the mixed solution into a die hole of a die by using a liquid transfer gun, freeze-drying the die filled with the mixed solution in a freeze dryer, and then demolding to obtain the freeze-dried patch. The die comprises a plate bottom, a die hole and a plate cover, wherein the plate bottom and the plate cover are connected in an inosculating manner, and the die hole is positioned on the plate bottom and is of a cylindrical structure; the diameter of the die hole is 25mm, and the height of the die hole is 5 mm; the bottom of the plate is provided with 50 die holes, and the prepared freeze-drying paste is a cylindrical tablet with the diameter of 25mm and the height of 2.5 mm. In addition, the steps of the lyophilization process are as follows: placing the mould filled with the mixed solution at the temperature of-15 ℃ for primary pre-freezing treatment, and then placing the mould at the temperature of-40 ℃ for secondary pre-freezing treatment; placing the mold after the secondary pre-freezing treatment at the temperature of 0 ℃ for primary drying treatment, and then placing the mold at the temperature of 20 ℃ for secondary drying treatment, wherein the specific process parameters are set as the following table 5:

TABLE 5

Phases	Temperature/. degree.C	Time/min	Duration/min	Vacuum degree/mbar
					One-time pre-freezing	-15	60	30	-
Secondary pre-freezing	-40	60	60	-
					Primary drying	0	60	600	0.25
Secondary drying	20	60	400	0.008

Experimental example:

in-vitro animal irritation experiments were carried out on the lyophilized patch prepared according to the preparation method provided in example 6 according to technical safety standards for cosmetics (2015 edition), and the method specifically includes the following steps:

(1) screening: the test animals are 2.5 kg-3.0 kg healthy adult New Zealand white rabbits, the animals are fed for 7 days under laboratory conditions, are fed by full-nutrition pellet feed, are fed freely, are drunk freely, and are subjected to sodium fluorescein examination within 24 hours before the test starts. Animals with eye irritation symptoms, corneal defects, and conjunctival damage were eliminated.

(2) Grouping: 7 test animals are screened out and marked (9-15) for testing the irritation of the freeze-dried patch.

(3) The operation is as follows: on the day of the test, the test rabbits were fixed, the lower left eyelid of the test rabbits was gently pulled open, and 0.1mL of the test substance (the test substance obtained after dissolving the lyophilized patch provided in example 6 above in sterile water) was dropped into the conjunctival sac to passively close the upper and lower eyelids for 1s, thereby preventing the test substance from being lost. The other eye was not treated for self-control. The results of the sodium fluorescein test before and after the test in 7 animals are shown in FIG. 1. FIG. 1A is a graph showing the results of sodium fluorescein test in both eyes of the test animals within 24 hours before the start of the test, all without damage; b to C in FIG. 1 are comparative images of the left eye (B) to which the test substance was dropped for 72 hours and the right eye (C) to which the test substance was not dropped, and there was no difference in visibility with naked eyes; in fig. 1, D is a result chart of sodium fluorescein test performed on the left eye of the test animal 72 hours after the test substance is dropped, and the test paper is not discolored, which proves that the left eye is not damaged after the test substance is dropped.

(4) Clinical examination and scoring: the eyes of the test animals were examined at 1h, 24h, 48h, 72h, and 4d and 7d after instillation of the test substance. If no irritation response occurred for 72h, the test was terminated and after the observation and recording for 72h, a further examination was carried out by adding dropwise sodium fluorescein. If corneal involvement or other ocular irritation is found and no recovery occurs within 7d, the observation time is extended to no longer than 21d to determine the reversibility or irreversibility of the lesion, and observation reports of 7d, 14d, and 21d are provided. In addition to observations of the cornea, iris, conjunctiva, other damaging effects should be recorded and reported. The score for eye irritation response should be recorded at each examination according to the scoring criteria for eye damage in table 6.

TABLE 6

Eye damage	Integration
		Cornea: without ulcer formation or turbidity	0
Cornea: diffuse or diffuse turbidity, clear and visible iris	1
		Cornea: the translucent area is easily distinguished and the iris is blurred	2
Cornea: a grayish white translucent region appears, the details of the iris are unclear, and the pupil is barely visible	3
		Cornea: opacity and no recognition of iris	4
Iris: is normal	0
		Iris: the deep wrinkles, congestion, swelling, and moderate congestion around the cornea	1
Iris: bleeding, visible damage to the naked eye, no response to light (or one of them)	2
		Iris: vascular normality	0
Iris: blood vessel congestion is bright red	1
		Iris: blood vessel congestion is deep red, and blood vessels are not easy to be distinguished	2
Iris: diffuse hyperemia with purplish red color	3
		Without edema	0
Minor edema (including instant membrane)	1
		Marked edema with partial eyelid eversion	2
Edema to near-semi-closure of eyelid	3
		Edema to greater closure of the eyelids	4

(5) And (4) evaluating the results: the eye stimulation intensity of the test object on the eyes is judged according to the eye stimulation response grading of table 7 by evaluating the highest integral mean value and the recovery time of the stimulation response of the cornea, the iris or the conjunctiva of the animal at observation points of 24h, 48h and 72h respectively after the test object is given.

TABLE 7

The results of the above irritation test are shown in table 8 below, where table 8 is the evaluation results of the highest integral mean value and the recovery time of the irritation response of the cornea, iris or conjunctiva of the rabbit at the observation time points of 24h, 48h and 72h, respectively, and the integral of the cornea, iris and conjunctiva is 0, which proves that the freeze-drying patch of the present invention has no irritation.

TABLE 8

Test items	24h	48h	72h
				Cornea	0	0	0
Iris (iris)	0	0	0
				Conjunctiva (conjunctiva)	0	0	0

Secondly, the freeze-dried paste prepared by the preparation method provided by the embodiment 6 is used for human body safety and efficacy experiments, and the method specifically comprises the following steps:

(1) the volunteers were recruited 38 in total, each of the volunteers dispensed 28 sheets of the lyophilized patch (test article) provided in example 6 of the present invention and 28 parts of activating solution (used as a solvent for the lyophilized patch, without any active ingredient), and the patch was used 1 time per day after cleansing face, and the patch was used for 28 days in total, while the other skin care habits were not changed.

(2) When the test article is issued, the volunteer is required to use the test article on the spot, and before and 0.5h after use, the German CK multifunctional skin tester is used for collecting the water content and melanin data and taking pictures. The data mean was recorded as D0 before use and D1 after 0.5 h.

(3) Skin texture data was collected and photographed with a german CK multifunctional skin tester 28 days after the use of the test article, as shown in fig. 2, wherein a and B in fig. 2 are skin condition diagrams of volunteers example 1 before (fig. 2A) and 28 days after (fig. 2B) the use of the freeze-dried patch of the present invention; fig. 2, C and D, are graphs of the skin condition of volunteer example 2 before application of the freeze-dried patch of the present invention (fig. 2C) and after application of the test article for 28 days (fig. 2D); as shown in FIG. 3, A, B is a graph comparing the effect of volunteer example 3 before application of the lyophilized patch of the present invention (FIG. 3A) and after application for 28 days (FIG. 3B); C. d is a graph comparing the effect of example 4 before application of the freeze-dried patch of the present invention (FIG. 3C) and after application for 28 days (FIG. 3D); E. f is a graph comparing the effect of example 5 before application of the freeze-dried patch of the present invention (FIG. 3E) and after application for 28 days (FIG. 3F). The mean of the data after 28 days of use was recorded as D2.

(4) During the period that the volunteers use the test articles, every volunteer is called back for one week, two weeks, three weeks and four weeks respectively, and consultations are carried out according to the safety, comfort and the like of the used products.

(5) Calculating the difference between the average values of the data items after 0.5h of use and before use (D1-D0), calculating the difference between the average values of the data items after 28 days of use and before use (D2-D0), and when the data difference is positive, calculating the improvement degree and the improvement population ratio. The improvement degree is data difference/D0 multiplied by 100%, and the proportion of the number of the improved people is the number of the people with each improvement degree/the total number multiplied by 100%.

The experimental results are as follows:

(1) overall evaluation results: including safety and efficacy evaluations, as shown in figure 4 and table 9 below.

TABLE 9

Test items	Number of effective persons tested	Improve the number of people	Improving the proportion of people
				Safety feature	38	38	100％
Comfort feeling	38	38	100％
				Degree of softness	38	34	89.5％
Degree of gloss	38	36	94.8％
				Wrinkle changes	38	30	78.9％

As can be seen from Table 9: the freeze-drying patch provided by the embodiment of the invention has no irritation and is comfortable to apply in the trial process of effective testing crowds, which shows that the freeze-drying patch has good safety and comfort; 89.5 percent of volunteers use the freeze-drying patch of the invention to increase the moisture content of the skin, and the softness is improved in time; 94.7 percent of volunteers use the skin-melanin-reducing gel provided by the invention to reduce the skin melanin, and the glossiness is improved in time; 78.9% of volunteers use the freeze-drying paste of the invention to remove dry lines and fine lines to different degrees and fade true lines.

(2) The results of the evaluation of skin softness (immediate effect) are shown in fig. 5 and table 10 below.

Watch 10

As can be seen from table 10: after 89.5% of volunteers use the freeze-drying patch, the skin moisture content values are improved to different degrees, and the skin moisture improvement degree of 63.2% of the volunteers is more than 20%, which shows that the freeze-drying patch disclosed by the invention has the effects of improving the skin moisture content and improving the skin softness.

(3) The results of skin gloss evaluation (immediate effect) are shown in fig. 6 and table 11 below.

TABLE 11

As can be seen from Table 11: after the freeze-drying patch disclosed by the invention is used, 94.8% of the skin melanin values of the volunteers are reduced to different degrees, the skin glossiness is obviously improved, and 63.2% of the skin melanin values of the volunteers are reduced by more than 10%, so that the freeze-drying patch disclosed by the invention has the effects of obviously reducing the skin melanin, brightening the skin color and improving the skin glossiness.

(4) The skin wrinkle evaluation results (long term effect) are shown in fig. 7 and table 12 below.

TABLE 12

As can be seen from table 12: after the freeze-drying patch is used for 28 days, 78.9% of skin wrinkles of the volunteers are faded to different degrees, 60.5% of fine lines of the volunteers are obviously faded, and 18.4% of deep wrinkles of the volunteers are lightened, so that the freeze-drying patch has a better effect on improving the skin wrinkles.

In conclusion, the freeze-dried patch provided by the embodiment of the invention has the effects of reducing wrinkles, improving skin glossiness and improving skin softness.

In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Claims

1. The freeze-dried plaster containing the stem cell exosomes is characterized by comprising the following components in parts by weight: 0.0005-0.005 part of umbilical cord mesenchymal stem cell exosome, 0.1-1.5 parts of sodium alginate, 0.05-1 part of trehalose, 0.05-0.2 part of hydrolyzed sodium hyaluronate and 97.295-99.7995 parts of deionized water.

2. The freeze-dried patch containing the stem cell exosomes according to claim 1, which is characterized by comprising the following components in parts by weight: 0.001-0.003 part of umbilical cord mesenchymal stem cell exosome, 0.8-1.2 parts of sodium alginate, 0.4-0.6 part of trehalose, 0.08-0.12 part of hydrolyzed sodium hyaluronate and 98.077-98.719 parts of deionized water.

3. The freeze-drying patch containing the stem cell exosomes according to claim 1 or 2, wherein the preparation method of the umbilical cord mesenchymal stem cell exosomes comprises the following steps:

4. A preparation method of the freeze-dried patch containing the stem cell exosomes according to any one of claims 1 to 3, characterized by comprising the following steps:

5. The method for preparing a lyophilized patch containing a stem cell exosome according to claim 4, wherein the step of placing the mold filled with the mixed solution in a vacuum freeze dryer for lyophilization treatment specifically comprises:

6. The preparation method of the lyophilized patch containing the exosome of the stem cell according to claim 5, characterized in that the mold filled with the mixed solution is placed at a temperature of-20 to-10 ℃ for primary pre-freezing treatment, and then placed at a temperature of-50 to-30 ℃ for secondary pre-freezing treatment, wherein the duration of the primary pre-freezing treatment is 20 to 50min, and the duration of the secondary pre-freezing treatment is 50 to 80 min.

7. The preparation method of the lyophilized patch containing the exosomes of the stem cells according to claim 5, characterized in that the step of placing the mold after the secondary pre-freezing treatment at a temperature of-5 to 5 ℃ for primary drying treatment and then placing the mold at a temperature of 10 to 30 ℃ for secondary drying treatment is carried out, wherein the duration of the primary drying treatment is 400 to 700min, and the duration of the secondary drying treatment is 200 to 500 min.

8. The preparation method of the freeze-dried patch containing the stem cell exosomes according to claim 5 or 7, characterized in that the mould after the secondary pre-freezing treatment is placed at a temperature of-5 to 5 ℃ for primary drying treatment, and then placed at a temperature of 10 to 30 ℃ for secondary drying treatment, wherein the vacuum degree of the primary drying treatment is 0.2 to 0.3mbar, and the vacuum degree of the secondary drying treatment is less than 0.01 mbar.

9. A freeze-dried patch prepared by the preparation method of any one of claims 4 to 8.

10. Use of a lyophilized patch according to any one of claims 1-3 and 9 for the preparation of a cosmetic product for skin care.