CN114712399B

CN114712399B - Exosome ointment composition, exosome ointment and preparation method and application thereof

Info

Publication number: CN114712399B
Application number: CN202210507177.5A
Authority: CN
Inventors: 张建民; 王靓
Original assignee: Guodian Beijing Medicine Technology Co ltd
Current assignee: Guodian Beijing Medicine Technology Co ltd
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-08-26
Anticipated expiration: 2042-05-11
Also published as: CN114712399A

Abstract

The application relates to the technical field of medicines, and particularly discloses an exosome ointment composition, an exosome ointment and a preparation method and application thereof. Based on the exosome ointment composition, the exosome ointment composition comprises exosomes, buffer solution, silk fibroin, salicyl alcohol, beta-sitosterol, oil transdermal absorbent and emulsifier. The exosome ointment is prepared from the exosome ointment composition. The preparation method of the exosome ointment comprises the following steps: s1: suspending the exosome in a buffer solution to obtain an exosome suspension; s2: mixing the exosome suspension obtained in the step S1 with the rest components to obtain a mixture; s3: emulsifying the mixture obtained in the step S2 to obtain the exosome ointment. The use of the exosome ointment as a medicament for treating or alleviating an inflammatory skin disease. The application improves the treatment effect on psoriasis.

Description

Exosome ointment composition, exosome ointment and preparation method and application thereof

Technical Field

The application relates to the technical field of medicines, in particular to an exosome ointment composition, an exosome ointment and a preparation method and application thereof.

Background

Psoriasis is a clinically common chronic inflammatory skin disease characterized by thickening of the epidermis, immune response and vascular proliferation. Psoriasis has a long course of disease, is stubborn in condition and tends to relapse easily, and is usually treated for life, so that physical and psychological health of patients is seriously affected. The etiology is unclear at present, and the etiology can be related to factors such as heredity, infection, immunity, endocrine and the like. The treatment is mainly carried out by local external application, such as vitamin D3, glucocorticoid, immunosuppressant, etc.; however, in practice, the efficacy of such drugs is limited.

Disclosure of Invention

In order to improve the treatment effect on psoriasis, the application provides an exosome ointment composition, an exosome ointment and a preparation method and application thereof.

In a first aspect, the application provides an exosome ointment composition, which adopts the following technical scheme:

an exosome ointment composition comprises the following components in percentage by mass based on the exosome ointment composition:

0.01-0.04% of exosome, 75.00-85.00% of buffer solution, 0.02-0.04% of silk fibroin, 0.01-0.03% of salicyl alcohol, 0.01-0.03% of beta-sitosterol, 15.00-20.00% of oil transdermal absorbent and 1.00-4.00% of emulsifier.

In some embodiments, the exosomes are 0.03-0.04%.

In some embodiments, the exosome is 0.04%.

In some embodiments, the silk fibroin is 0.02%.

In some embodiments, the salicyl alcohol is 0.02%.

In some embodiments, the beta-sitosterol is 0.02%.

In some embodiments, the exosomes are 0.04%, the silk fibroin is 0.03%, the saligenin is 0.02%, and the beta-sitosterol is 0.02%.

In some embodiments, the exosome is obtained from stem cells by culturing and isolating. The stem cell is selected from the group consisting of an induced pluripotent stem cell and a mesenchymal stem cell.

In some embodiments, the buffer is selected from the group consisting of normal saline, PBS buffer, and DPBS buffer. Preferably, the buffer is a DPBS buffer.

In some embodiments, the lipid based transdermal absorption agent is selected from the group consisting of olive oil, grape seed oil, tea seed oil, shea butter, almond oil, and jojoba oil.

In some embodiments, the emulsifier is selected from the group consisting of SEPIPLUS 400 emulsifier, SIMULGEL 600 emulsifier, and Montanov 68 emulsifier.

In a second aspect, the application provides an exosome ointment, which adopts the following technical scheme:

an exosome ointment is prepared from the exosome ointment composition.

In a third aspect, the application provides a preparation method of an exosome ointment, which adopts the following technical scheme:

a preparation method of an exosome ointment comprises the following steps:

s1: suspending the exosome in a buffer solution to obtain an exosome suspension;

s2: mixing the exosome suspension obtained in the step S1 with the rest components to obtain a mixture;

s3: emulsifying the mixture obtained in the step S2 to obtain the exosome ointment.

In a third aspect, the present application provides a use of an exosome ointment as a medicament for treating or alleviating an inflammatory skin disease.

In some embodiments, the inflammatory skin disease includes, but is not limited to, psoriasis.

In some embodiments, the skin affliction with the immune-related disorder includes, but is not limited to, allergic dermatitis, atopic dermatitis, dermatomyositis, eczema, acne, sjogren's syndrome, systemic lupus erythematosus, behcet's disease, systemic sclerosis.

In summary, the present application has the following beneficial effects:

the exosome ointment therapy provided herein is able to alleviate the symptoms of psoriasis. The exosome plays a key role in improving the psoriasis treatment effect of the exosome ointment; the presence of silk fibroin, saligenin and beta-sitosterol plays a secondary role in improving the psoriasis treatment effect of the exosome ointment, and the silk fibroin, saligenin and beta-sitosterol can synergistically improve the treatment effect of the exosome ointment.

Drawings

FIG. 1 is a photograph of a depilated area of a control group of mice at day 3;

FIG. 2 is a photograph of a depilated area of a control group of mice at day 9;

FIG. 3 is a photograph of the state of the mouse depilated area at day 3 of the experimental group (example 4);

FIG. 4 is a photograph showing the state of the mouse depilation region on day 9 in the experimental group (example 4);

FIG. 5 is a photograph of the state of the mouse depilated area at day 3 of the experimental group (comparative example 6);

fig. 6 is a photograph of the state of the mouse depilated area at day 9 of the experimental group (comparative example 6);

FIG. 7 is the HE staining of pathological sections of mouse depilated areas on day 10 in the control group;

FIG. 8 is the results of HE staining of pathological sections of mouse depilated areas at day 10 in the experimental group (example 4);

FIG. 9 is the HE staining result of pathological sections of mouse depilated area at day 10 in the experimental group (comparative example 6);

FIG. 10 is the concentration of the inflammatory factor IL-17A in the skin lesion tissue of the mice of the control group, the experimental group (example 4 and comparative example 6);

FIG. 11 is the concentration of the inflammatory factor IL-10 in the skin lesion tissues of the mice of the control group, the experimental group (example 4 and comparative example 6);

FIG. 12 is the concentration of the inflammatory factor TGF-. beta.in the skin lesion tissues of the mice of the control group, experimental group (example 4 and comparative example 6);

FIG. 13 is the concentration of the inflammatory factor IL-1. beta. in the skin lesion tissue of the mice of the control group, the experimental group (example 4 and comparative example 6);

FIG. 14 is a graph showing the concentration of inflammatory factor TNF-. alpha.in skin lesion tissues of mice of the control group and the experimental group (example 4 and comparative example 6).

Detailed Description

The present application will be described in further detail with reference to the following drawings and examples.

The source of the exosome is national classic (Beijing) pharmaceutical technology Co., Ltd

A preparation method of an exosome ointment comprises the following steps:

Examples 1 to 10 and comparative examples 1 to 6

TABLE 1 ingredient tables (by weight) of exosome ointments of examples 1 to 10 and comparative examples 1 to 6

Remarking: "-" represents the addition amount of 0.

Mouse assay

Imiquimod (IMQ) can induce the maturation and secretion of dendritic cells in a mouse body and further activate the activation of T lymphocytes around skin lesions, so that the autoimmune reaction is caused, and finally, the pathological changes such as erythema, scale, skin lesion infiltration hypertrophy and the like are caused, and an ideal animal model is provided for the evaluation of the drug effect of the psoriasis medicament.

The experimental method comprises the following steps: Balb/C mice (male, 7-8 weeks old, 18-20 g) were used in the experiment and divided into 3 groups of 6 mice each. The experimental mice were divided into control group, model group, experimental group, and the number of mice per group was 6.

The mice were depilated on their backs, and the dimensions of the depilated area were 2cm by 2.5 cm. The hair on the back of the mouse was shaved using a shaver and a depilatory cream, and then the depilatory cream and hair remaining on the back of the mouse were wiped off using a cotton ball soaked with physiological saline, and the skin on the back of the mouse was visually observed to be pink.

Control group: vaseline is applied to the depilated area of the mouse every day for 1 time every day, and the application dose is 50 mg/mouse. Mice were sacrificed on day 10 and the tissues were harvested for skin lesions.

Experimental groups: on days 0 to 9, the mice were applied with 5% imiquimod ointment (Sichuan mingxin pharmaceutical Co., Ltd.) per day 1 time per day at a dose of 50 mg/mouse/application. Starting from day 2, after applying 5% imiquimod ointment for 2 hours, the mouse depilatory area was applied with exosome ointment (examples 1-10 and comparative examples 1-6) at a dose of 5 mg/mouse/application. Mice were sacrificed on day 10 and the tissues were harvested for skin lesions.

The control and experimental groups were scored at day 9 with reference to Psoriasis PASI (Psoriasis area and severity index) scoring criteria, resulting in cumulative scores as shown in table 2. Mice were assessed at day 9 for severity of skin inflammation including skin thickness, scabbing, and erythema on the depilated area and scored separately using a 5 point scale (0-4) with each mouse scored separately, with each group being the average of all mice scored within the group.

Skin thickness was scored as:

0: smooth and wrinkle-free skin

1: slight wrinkles appeared on the skin at the edges of the application area

2: slight wrinkles appeared on the skin in the application area

3: the wrinkle degree of the medicine coating area is further deepened

4: on the basis of the score of 3, the mice have the conditions of weight loss, poor state and the like

The scoring criteria for scab were:

0: smooth and non-scaling skin

1: slight scaling of the skin in the application area

2: the whole skin of the application region is covered with scales

3: the scale degree of the medicine coating area is further deepened

The skin erythema scoring criteria were:

0: the skin is smooth.

1: the skin appeared slightly red in the area of application

2: the skin of the application area turns red

3: the red color of the medicine coating area is further deepened

Cumulative score for each group of mice: sum of three scores for skin thickness, scab and skin erythema.

TABLE 2 cumulative psoriasis PASI scores for control and experimental groups

As can be seen from table 2, the exosome ointment treatment provided herein is able to alleviate the symptoms of psoriasis.

As can be seen from comparison of examples 1 to 4, the exosome ointment is more effective in treating psoriasis symptoms as the amount of exosome used increases.

As can be seen by comparing example 4 with comparative example 1, exosomes play a key role in enhancing the psoriasis treatment effect of exosome ointments. Further, it can be seen from comparing example 4 and comparative example 2 that the presence of silk fibroin, saligenin, and β -sitosterol play a secondary role in enhancing the psoriasis treatment effect of the exosome ointment. Furthermore, it can be seen from comparison of example 4 and comparative examples 2 to 5 that silk fibroin, saligenin, and β -sitosterol can synergistically enhance the therapeutic effect of an exosome ointment.

Photographs of the depilated areas on the backs of mice of the control group and the experimental group (example 4 and comparative example 6) were taken on the 3 rd day and the 9 th day, respectively, and photographs of the depilated areas on the 3 rd day and the 9 th day of the mice of the control group and the experimental group (example 4 and comparative example 6) were shown in fig. 1 to 6. Therefore, the conditions of the squamous folds and the like on the back of the mice treated by the exosome ointment provided in the example 4 are obviously better than the conditions of the squamous folds and the like on the back of the mice treated by the exosome ointment provided in the comparative example 6, and the treatment of the exosome ointment can relieve the psoriasis symptoms.

On day 10, three mice were randomly selected from the control group and the experimental group (example 4 and comparative example 6), skin tissues of the depilated area on the back of the selected mice were completely peeled off, and the cut-out portions were paraffin-embedded and used for HE staining of pathological sections to evaluate pathological indexes. The pathological section HE staining results are shown in FIGS. 7 to 9. Therefore, the control group has thin skin and normal cell morphology; the skin lesion acanthocyte parakeratosis of the experimental group (comparative example 6), acanthosis hypertrophy, inflammatory cell infiltration around the dermal superficial blood vessel, and typical phenotype of psoriasis sample; the experimental group (example 4) significantly reduced the thickness of the epidermal layer, reducing the IMQ-induced psoriasis phenotype. It can be shown that the treatment of the exosome ointment provided by the present application can alleviate the symptoms of psoriasis.

Then, the cut part was used for MSD detection of factor concentration in the skin lesion tissue, and the results are shown in FIGS. 10 to 14. Therefore, the exosome ointment can obviously reduce the levels of inflammatory factors IL-17A, IL-10, TGF-beta, IL-1 beta and TNF-alpha in skin lesion tissues of psoriasis model mice.

It will be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not limited thereto. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and substance of the invention, and these modifications and improvements are also considered to be within the scope of the invention.

Claims

1. An exosome ointment composition, characterized in that, on the basis of the exosome ointment composition,

the exosome ointment composition comprises the following components in percentage by mass: exosome 0.01%, DPBS buffer 78.92%, silk fibroin 0.03%, salicyl alcohol 0.02%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.02%, DPBS buffer 78.91%, silk fibroin 0.03%, salicyl alcohol 0.02%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.03%, DPBS buffer solution 78.90%, silk fibroin 0.03%, salicyl alcohol 0.02%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.04%, DPBS buffer 78.89%, silk fibroin 0.03%, salicyl alcohol 0.02%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.04%, DPBS buffer solution 78.9%, silk fibroin 0.02%, salicyl alcohol 0.02%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.04%, DPBS buffer 78.88%, silk fibroin 0.04%, salicyl alcohol 0.02%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.04%, DPBS buffer solution 78.9%, silk fibroin 0.03%, salicyl alcohol 0.01%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.04%, DPBS buffer 78.88%, silk fibroin 0.03%, salicyl alcohol 0.03%, beta-sitosterol 0.02%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.04%, DPBS buffer solution 78.9%, silk fibroin 0.03%, salicyl alcohol 0.02%, beta-sitosterol 0.01%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

or the exosome ointment composition comprises the following components in percentage by mass: exosome 0.04%, DPBS buffer 78.88%, silk fibroin 0.03%, salicyl alcohol 0.02%, beta-sitosterol 0.03%, olive oil 18.00%, SEPIPLUS 400 emulsifier 3.00%;

the exosome is obtained by culturing and separating stem cells, and the stem cells are induced pluripotent stem cells or mesenchymal stem cells.

2. An exosome ointment prepared from the exosome ointment composition of claim 1.

3. A method of preparing an exosome ointment according to claim 2, comprising the steps of:

4. Use of an exosome ointment according to claim 2 in the preparation of a medicament for treating or alleviating an inflammatory skin disease, an immune-related disease, or a cutaneous involvement.

5. Use according to claim 4, characterized in that said inflammatory skin diseases include, but are not limited to, psoriasis.