CN106620815A - Gel dressing based on G-quadruplex/collagen, as well as preparation and application of gel dressing - Google Patents

Abstract

The invention discloses a gel dressing based on G-quadruplex/collagen and a preparation method thereof. Guanine nucleoside, boric acid and potassium hydroxide are added into deionized water at a ratio of 1 to 3:1:1, and heated to 90-98°C to obtain G-quadruplex solution; cool to 35-40°C, add collagen to disperse it so that the concentration of collagen in the solution is 2-10 mg/mL, and cool to room temperature to obtain the G‑tetrad/collagen gel dressing. The preparation method has simple reaction conditions, few synthesis steps, short time consumption and low production cost. When the gel dressing is used for wound healing, it has good biocompatibility, good air permeability, strong adhesion, high water content, maintains a humid environment, and makes the wound difficult to scab. The gel dressing of the invention does not contain any irritating ingredients and has no toxic and side effects. The gel dressing is used to accelerate wound healing and has potential application value.

Description

G-tetrad/collagen based gel dressing and its preparation and application

technical field

The invention belongs to the field of medical dressings, and relates to a gel dressing and a preparation method thereof, in particular to a G-quadruplex/collagen-based gel dressing capable of promoting wound healing and a preparation method thereof.

Background technique

Wound is the damage of normal skin caused by external factors such as surgery, heat, external force, electric current, low temperature, etc. If left untreated, the wound may become infected or worsen. Traditionally used dressings are gauze, cotton and other materials, but the effects of these dressings on hemostasis, moisturizing, preventing wound infection and promoting wound healing are not satisfactory.

Collagen is the main component of animal connective tissue and the most abundant functional protein in mammals. Collagen has been widely used as a biomaterial due to its low immunogenicity, degradability, biocompatibility, and coagulation properties. Compared with other wound dressings, the collagen-containing gel dressing is easy to prepare, takes a short time, has good biological activity, and has a good effect on wound healing.

The existing gel dressing mainly also has the following problems: 1. In terms of preparation, the synthesis of the gel also has deficiencies such as complicated preparation and long time consumption. II. In terms of performance, the gel has poor mechanical strength and has certain biological toxicity. III In terms of wound healing, some dressings have poor air permeability and long wound healing time. Therefore, preparing a simple and effective gel dressing for rapid wound healing will have important research significance and application value.

Contents of the invention

Aiming at the above-mentioned deficiencies of the prior art, the object of the present invention is to solve the complicated problem of preparation of the gel dressing, overcome the deficiencies of the gel dressing such as poor air permeability, long healing time, and poor biocompatibility; select degradable, biocompatible Using G-quadruplex and collagen as raw materials, the prepared G-quadruplex/collagen-based gel dressing has good mechanical properties, keeps the wound moisturized, adheres and avoids infection, effectively, Promotes rapid wound healing.

The present invention prepares the reaction mechanism of the G-quadruplex/collagen gel dressing that can promote wound healing as shown in Figure 1:

Guanine nucleosides are combined with adjacent guanosine nucleosides due to hydrogen bonds, and the four connected guanosine nucleosides coordinate with a K+ to form a G-quadruplex; G-quadruplexes are connected by The borate groups are bridged together to form a spatial network. When collagen is added, the collagen becomes embedded in it, eventually forming a G-quadruplex/collagen based gel.

According to above-mentioned principle, the present invention adopts following technical scheme:

A preparation method based on G-quadruplex/collagen gel dressing, comprising the following steps:

(1) Add guanosine, boric acid and potassium hydroxide into water, heat, and wait for the guanosine to be completely dissolved to obtain a G-quadruplex solution;

(2) After cooling the G-quadruplex solution in step (1), add collagen to disperse the collagen to obtain the G-quadruplex/collagen-based gel dressing.

Wherein, in the step (1),

The guanosine can also be replaced by adenosine and cytosine.

Described potassium hydroxide can also be replaced with sodium hydroxide, lithium hydroxide.

The molar ratio of guanosine, boric acid and potassium hydroxide is 1-3:1:1; preferably, 2:1:1.

The water is deionized water.

The heating temperature is 90-98°C; preferably 95°C.

The heating time is 15-25 minutes; preferably 15-20 minutes.

Wherein, in the step (2),

The cooling temperature is 35-40°C.

The collagen can be from but not limited to tilapia skin, cod skin.

The concentration of the dispersed collagen is 2-10 mg/mL; preferably 5 mg/mL.

Specifically, the steps include:

(1) Add guanosine, boric acid and potassium hydroxide into deionized water at a molar ratio of 1 to 3:1:1, shake for 5 to 10 minutes, heat the water bath to 90 to 98°C, and keep a constant temperature of 15 to 25 Minutes, until the guanosine is completely dissolved, and the G-quadruplex is obtained.

(2) Slowly cool the solution in the above step (1) to 35-40°C, keep the water bath at a constant temperature, add collagen so that the concentration of collagen in the solution is 2-10mg/mL, shake it to disperse quickly, After cooling to room temperature, a G-quartet/collagen based gel dressing was prepared.

Wherein, in step (1), after adding guanosine, first add boric acid, then add potassium hydroxide, and after adding boric acid or potassium hydroxide, shake rapidly for 20 seconds, which helps the dissolution of guanosine.

Wherein, in step (1), the molar ratio of guanosine, boric acid and potassium hydroxide is 2:1:1, and the water bath heating temperature is 95° C. The reaction conditions are the best; the concentration of collagen in the above step (2) The best dressing effect is 5mg/mL.

The present invention also proposes the gel dressing based on G-quadruplex/collagen prepared by the above preparation method.

The G-tetrad/collagen-based gel dressing of the present invention has simple composition, convenient preparation, good air permeability, strong adhesion, good mechanical properties and good biocompatibility.

The present invention also proposes the application of the gel dressing based on G-quadruplex/collagen in promoting the proliferation and differentiation of cells in wounds.

The present invention also proposes the application of the G-quadruplex/collagen-based gel dressing in the preparation of products for promoting wound healing.

In the present invention, the G-quadruplex/collagen-based gel dressing can be used as a biomaterial.

In the present invention, the G-quadruplex/collagen-based gel dressing can accelerate wound healing.

In the present invention, the gel dressing based on G-quadruplex/collagen is used for wound healing in mice, and has the following characteristics: 1. fast hemostasis, absorption of tissue secretions, and enhanced adhesion in the wound; 2. maintenance of wound relative A humid environment reduces the chance of infection; ③ accelerates cell proliferation and differentiation, and promotes skin regeneration.

In the present invention, the G-quadruplex/collagen-based gel dressing includes, but is not limited to, a wound dressing.

In the present invention, the G-quadruplex/collagen-based gel dressing includes, but is not limited to, used for wound healing in mice.

In the present invention, the G-quartet/collagen-based gel dressing includes, but is not limited to, the loading of drugs.

The preparation method of the G-quadruplex/collagen-based gel dressing provided by the invention has simple reaction conditions, few synthesis steps, short time consumption and low production cost. The G-quadruplex/collagen gel dressing provided by the present invention is used for wound healing, has the advantages of good biocompatibility, good air permeability, strong adhesion, and high water content, and maintains a moist environment for the wound, enabling The wound is not easy to scab. The G-quadruplex/collagen-based gel dressing of the present invention does not contain any irritating ingredients and has no toxic and side effects. The G-quadruplex/collagen-based gel dressing provided by the invention is used for accelerating wound healing and has potential application value.

Description of drawings

Fig. 1 is a diagram showing the reaction mechanism of the present invention to prepare G-quadruplex/collagen-based gel dressing.

Figure 2 is the gel prepared in Example 2.

Fig. 3 is the healing situation of the wounds of the control group and the treatment group (gel prepared in Example 2) on days 0, 3, 5, and 7.

Fig. 4 shows the differences in wound area between the control group and the treatment group (gel prepared in Example 2) on days 0, 3, 5, and 7.

Fig. 5 is the histological situation of the control group and the treatment group (the gel prepared in Example 2) on the 3rd, 5th and 7th day.

Fig. 6 is the cytotoxicity experiment of the control group and the treatment group (the gel prepared in Example 2).

detailed description

The present invention will be further described in detail in conjunction with the following specific embodiments and accompanying drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the content specifically mentioned below, are common knowledge and common knowledge in this field, and the present invention has no special limitation content.

Example 1

(1) Add guanosine, boric acid and potassium hydroxide into deionized water at a molar ratio of 1:1:1, shake for 5 minutes, heat the water bath to 90°C, and keep the temperature for 20 minutes until the guanosine is completely dissolved .

(2) Slowly cool the above solution to 37°C, keep the water bath at a constant temperature, add collagen so that the concentration of collagen in the solution is 5 mg/mL, shake it to disperse it quickly, and cool it to room temperature to obtain a G-quadruple body/collagen gel dressing.

Example 2

(1) Add guanosine, boric acid and potassium hydroxide into deionized water at a molar ratio of 2:1:1, shake for 10 minutes, heat the water bath to 95°C, and keep the constant temperature for 15 minutes until the guanosine is completely dissolve.

(2) Slowly cool the above solution to 40°C, keep the water bath at a constant temperature, add collagen to make the concentration of collagen in the solution 7mg/mL, shake it to make it disperse quickly, cool to room temperature, and prepare the product based on G - Gel dressing of tetrad/collagen. The prepared gel is shown in Figure 2. When the prepared gel is placed upside down, the gel will stick to the bottle wall, unlike the aqueous solution that will flow down; and after 15 days, the gel will still stick to the bottle wall when placed upside down. This illustrates the good mechanical properties of the G-tetrad/collagen based gel dressing.

Example 3:

The gel prepared in Experimental Example 2 was used for wound healing of mice as a treatment group.

In this experiment, a traumatic scar model was made on the back of ICR mice by surgical method. By applying the gel on the surface of the wound, observe the healing of the surface scars every day, and evaluate the repairing effect of the product on the scars.

(1) modeling

Under anesthesia by intraperitoneal injection of 1% pentobarbital sodium 5mL/kg, the back of the mouse was sheared, depilated with 4% sodium sulfide, and a circular incision with a diameter of about 1.5 cm was made on both sides of the parallel spine. If there is slight bleeding, wipe the mouse with a sterile cotton ball to stop the bleeding, and return it to the mouse box.

(2) Animal grouping

The successfully modeled ICR mice were randomly divided into groups a, b, and c, with 3 mice in each group.

A control group: the wound on the right side of the mouse was used as the control group.

B treatment group: the wound on the left side of the mice was used as the treatment group.

The control group was allowed to heal normally without any treatment. The gel dressing was applied to the wounds of the treatment group once a day, and the surface scar repair was observed every day. When the experiment was carried out to the third day, the mice in group a were treated, and the wounds were sliced for histological characterization, and the growth of wound surface cells was observed. When the experiment was carried out to the fifth day, the mice in group b were treated, and the wounds were sliced for histological characterization, and the growth of wound surface cells was observed. When the experiment was carried out to the 7th day, the mice in group c were treated, and the wounds were sliced for histological characterization, and the growth of wound surface cells was observed.

(3) Results

As shown in Figure 3, when the gel dressing is applied to the mouse wound, the dressing will adhere to the wound and will not fall off like a liquid, which shows that the dressing has strong adhesion; after 4 hours, the dressing will dry, which shows the air permeability it is good. On the 3rd day after the operation, the mouse wounds contracted and scabbed to a certain extent, but it was obvious that there was a significant difference in the wound healing between the control group and the treatment group. Because, the scar healing rate of the gel dressing smear treatment to the treatment group is significantly better than that of the control group. By the 7th day, the wound area of the mice in the treatment group was extremely small, basically healed completely, and the recovery effect was good.

As shown in Figure 4, through the comparison of the wound area of the control group and the treatment group at different time periods, it was clearly observed that the wound healing rate of the treatment group was far better than that of the control group, indicating that the gel dressing based on G-quadruplex/collagen Good for wound healing.

As shown in Figure 5, since the gel dressing was applied to the treatment group, collagen promoted the proliferation and differentiation of cells in the wound. From the histological observation, the degree of cell epithelialization in the treatment group was better than that in the control group, which promoted the skin regeneration of the mice in the treatment group significantly faster than that in the control group. By day 7, the skin of mice in the treatment group had basically regenerated completely.

Example 4 Cytotoxicity Experiment

The gel prepared in Experimental Example 2 was filtered with a 0.22 μm sterile filter membrane to remove bacteria in the gel. The sterilized gel was used in the fibroblast cytotoxicity experiment as the treatment group; the blank was used as the control group.

In this experiment, MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) method was used to measure cell viability. The biocompatibility of the gel was evaluated by measuring the toxicity of the gel to cells.

(1) Fill the edge wells of a 96-well plate with sterile PBS; collect logarithmic phase cells, adjust the cell suspension concentration to 50,000 cells/mL, and add 100 μL of cell suspension to each well.

(2) 5% CO ₂ , incubate at 37°C until the cell monolayer covers the bottom of the well, then add 10 μL of 5 mg/mL gel.

(3) The cells were placed in an incubator, incubated at 37° C. for 24 hours in 5% CO ₂ , and observed under an inverted microscope.

(4) Add 20 μL of 5 mg/mL MTT to each well, and continue culturing for 4 h.

(5) Terminate the culture, suck off the culture medium in the wells; add 150 μL dimethyl sulfoxide to each well, shake the shaker at low speed for 10 minutes, and then use a microplate reader to detect the absorbance value of each well at OD490nm.

(6) At the same time, set the zero adjustment well (medium, MTT, dimethyl sulfoxide) and the control well (cells, gel with the same concentration, medium, MTT, dimethyl sulfoxide).

(7) Calculation of cell viability:

Cell survival rate=(A value of gel group-A value of zero-adjusted well)/(A value of control well-A value of zero-adjusted well)×100%

As shown in Figure 6, the cytotoxicity experiments were performed with fibroblasts. Experimental results show that: the cell survival rates of both the control group and the treatment group are close to 100%. This shows that the gel dressing has very little toxicity to fibroblasts, which means that the gel dressing has good biocompatibility.

The foregoing are only a few specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principle of the present invention, the use of G-quadruplex/collagen-based gel dressing obtained by the same or similar method for wound healing is within the protection scope of the present invention.

Claims (10)

1. a preparation method based on the gel dressing of G-quadruplex/collagen, it is characterized in that, the concrete steps of this method are: (1) adding guanosine, boric acid and potassium hydroxide into water, heating, and after guanosine is completely dissolved, a G-quadruplex solution is obtained; (2) After the G-quadruplex solution in step (1) is cooled, collagen is added to disperse the collagen to obtain the G-quadruplex/collagen-based gel dressing. 2. the preparation method as claimed in claim 1, is characterized in that, in described step (1), described guanosine can also be replaced with ... adenosine, cytosine nucleoside; Described potassium hydroxide Sodium hydroxide and lithium hydroxide can also be used instead. 3. The preparation method according to claim 1, characterized in that, in the step (1), the molar ratio of the guanosine, boric acid and potassium hydroxide is 1-3:1:1. 4. The preparation method according to claim 1, characterized in that, in the step (1), the heating temperature is 90-98° C.; the heating time is 15-25 minutes. 5. The preparation method according to claim 1, characterized in that, in the step (2), the cooling temperature is 35-40°C. 6. the preparation method as claimed in claim 1 is characterized in that, in described step (2), described collagen is from tilapia fish skin, cod fish skin. 7. The preparation method according to claim 1, characterized in that, in the step (2), the concentration of the dispersed collagen is 2-10 mg/mL. 8. The gel dressing based on G-quadruplex/collagen prepared by the preparation method according to claim 1. 9. the application of the gel dressing based on G-quadruplex/collagen as claimed in claim 8 in the product that promotes cell proliferation, differentiation. 10. The application of the gel dressing based on G-quadruplex/collagen as claimed in claim 8 in the preparation of products for promoting wound healing.