Skin damage repair gel patch and its preparation method and application

Abstract

The present invention belongs to the field of skin repair, and particularly relates to a skin injury repair gel patch, a preparation method thereof and an application. The preparation method adopts the following steps: (1) preparing an aqueous alginate solution; (2) adding free radical scavenging molecules into an aqueous hyaluronic acid solution; (3) adding the free radical scavenging molecules coated with hyaluronic acid into the aqueous alginate solution and adding a crosslinking agent for crosslinking; pouring the mixture into a mold or loading it on a gauze or other fibers to form a skin injury repair gel patch. By adding anti-inflammatory molecules with amino groups to regulate the composition of alginate, and at the same time wrapping hydrophobic free radical scavenging factors in a transdermal absorbable hyaluronic acid solution, the present invention realizes the rapid release of hydrophobic free radical scavenging factors. At the same time, the hydrogel can also block the invasion of external bacteria and avoid the occurrence of skin ulceration caused by bacterial infection on the wound surface.

Description

Technical Field

[0001] The present invention belongs to the field of skin repair, and particularly relates to a skin injury repair gel patch, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrogel is a porous material with a three-dimensional network structure, having good hydrophilicity and not dissolving, integrating properties such as moisture retention and water retention, and is a type of material commonly used in tissue engineering repair. Alginate is a natural polysaccharide with good biocompatibility and is widely used in the field of tissue engineering. However, negatively charged alginate will cause an immune rejection effect when contacting the body. Free radical scavenging factors are mostly hydrophobic drugs, which are difficult to maintain on the wound surface for a long time when directly applied, and there is a potential risk of cytotoxicity for a large amount of drugs at the injury site. Therefore, how to achieve the slow release of hydrophobic drugs in the hydrogel, while scavenging free radicals and promoting angiogenesis at the injury site is an urgent problem to be solved.

[0003] With the continuous progress of science and technology, nuclear technology is increasingly used in daily life and production. While nuclear technology brings many conveniences, exposure to nuclear radiation due to accidents or improper operations also causes certain damage to people's daily life. Not only nuclear radiation and radiotherapy, but also skin damage caused by solar ultraviolet radiation in daily life requires care and treatment. Ionizing radiation is mainly due to the fact that after the body is irradiated, the biological macromolecules, proteins, etc. in the body change in morphology and structure due to energy transfer. Since the human body contains more than 70% water, the reactive oxygen species and free radicals generated after being irradiated by ionizing radiation are the main causes of radiation damage, seriously affecting human health. Different from ordinary skin injuries (such as cuts, contusions, full-thickness skin injuries), irradiated skin does not show obvious injuries, but the DNA of deep skin cells will break due to radiation energy, and the broken DNA will form deformed cells during self-replication. At the same time, a large number of free radicals are generated during the radiation process, resulting in cell death due to oxidative stress and subcutaneous vascular malformation, which are the main reasons why irradiated skin is more difficult to repair. Therefore, quickly scavenging free radicals and promoting blood vessel regeneration at the injury site are very important for promoting skin radiation injury. However, the existing clinical treatment methods for radiation-induced injuries mostly use surgical debridement, hyperbaric oxygen therapy, etc., which are complex in operation and expensive. Summary of the Invention

[0004] The purpose of the present invention is to provide a skin injury repair gel patch, a preparation method thereof, and an application thereof.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A preparation method of a skin injury repair gel patch, adopting the following steps:

[0007] (1) Prepare an aqueous alginate solution, stir overnight, and fully swell.

[0008] (2) Add radical scavenging molecules to the aqueous hyaluronic acid solution, stir at room temperature, and centrifuge to discard the excess hyaluronic acid to obtain radical scavenging molecules coated with hyaluronic acid.

[0009] (3) Add the radical scavenging molecules coated with hyaluronic acid obtained in (2) above to the aqueous alginate solution in step (1), and mix well to obtain a mixed solution.

[0010] (4) Add a crosslinking agent to the mixed solution obtained in step (3) for crosslinking.

[0011] (5) Pour the mixed solution added with the crosslinking agent in step (4) into a mold or load it on a gauze or other fiber to form a skin injury repair gel dressing.

[0012] The radical scavenging molecules are one or more of astaxanthin, resveratrol, curcumin, gallic acid, ferulic acid, dopamine, and the concentration of the radical scavenging molecules is 0.005 - 0.02 (g / ml) of the volume of the hyaluronic acid solution.

[0013] The mass - volume concentration of the alginate is 0.01 - 0.02 (g / ml), and the molecular weight of the alginate is 10 kDa - 500 kDa.

[0014] The mass - volume concentration of the aqueous hyaluronic acid solution is 0.01 - 0.02 (g / ml), and the molecular weight is 2 kDa - 500 kDa.

[0015] The crosslinking agent is one or a mixture of two of polymer molecules with amino groups and divalent metal ions.

[0016] The polymer molecules with amino groups are one or a mixture of more of quaternized chitosan, chitosan oligosaccharide, and L - arginine.

[0017] The divalent metal ion is one of calcium, magnesium, copper, and barium.

[0018] Preferably, the preparation method of the skin injury repair gel dressing includes the following steps:

[0019] (1) Weigh sodium alginate powder, swell it with deionized water overnight to obtain a uniform and transparent aqueous sodium alginate solution with a concentration of 0.01 g / ml.

[0020] (2) Weigh hyaluronic acid powder, swell it in deionized water overnight to obtain a hyaluronic acid solution with a concentration of 0.01 g / ml.

[0021] (3) Add the free radical scavenging factor to the hyaluronic acid solution, and stir at high speed until uniform to form a free radical scavenging factor sodium hyaluronate solution with a concentration of (7.5-10) / 0.5 (mg / ml). After centrifugation, add this solution to the sodium alginate solution at a volume ratio of 1:1-2, and stir at high speed to form a uniform mixed solution of sodium alginate and hyaluronic acid solution;

[0022] (4) Add a 1% mass fraction quaternized chitosan solution and a 10% mass fraction divalent metal ion solution to the mixed solution of sodium alginate solution and hyaluronic acid solution, and achieve molecular cross-linking through electrostatic interaction; wherein, the volume ratio of the quaternized chitosan solution to the divalent metal ion solution is 1:5;

[0023] (5) Pour the mixed solution of the quaternized chitosan solution and the divalent metal ion solution added in step (4) into a mold or load it on a gauze or other fibers to form a skin injury repair gel containing the functions of scavenging free radicals and antibacterial.

[0024] The present invention also includes a skin injury repair gel dressing obtained by the above preparation method.

[0025] The present invention also includes an application of the skin injury repair gel dressing, which is applied to the preparation of a dressing for skin injuries.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] The present invention adjusts the composition of sodium alginate by adding an anti-inflammatory molecule with an amino group, and at the same time wraps the hydrophobic free radical scavenging factor in a transdermal absorbable hyaluronic acid solution to achieve rapid release of the hydrophobic free radical scavenging factor. This hydrogel material based on radiation damage can provide a moist environment for the damaged area, and at the same time has the dual functions of scavenging free radicals caused by radiation and promoting angiogenesis, especially suitable for delaying the occurrence of radiation damage; at the same time, the hydrogel can also block the invasion of external bacteria and avoid skin ulceration caused by bacterial infection on the wound surface.

[0028] At the same time, the matrix materials used in the present invention, sodium alginate and hyaluronic acid, are both natural polymers, with good biocompatibility, mild preparation conditions, simple operation, no by-products, no toxicity, and no need to add catalysts; the skin injury repair gel dressing provided by the present invention has a certain preventive and therapeutic effect on solar ultraviolet radiation skin injury, skin radiation injury caused by nuclear radiation or nuclear contamination, and skin injury of radiotherapy patients. Description of the Drawings

[0029] Figure 1 It is a morphology diagram of skin injury repair gel dressings with different sizes prepared in Examples 1-3 of the present invention.

[0030] Figure 2 The water content of the skin injury repair gel patch prepared in Examples 1-3 of the present invention.

[0031] Figure 3 The biocompatibility characterization results of the skin injury repair gel patch prepared in Examples 1-3 of the present invention. Figure 4 The free radical scavenging effect characterization results of the skin injury repair gel patch prepared in Examples 1-3 of the present invention.

[0032] Figure 5 The effect diagram of Example 3 of the present invention for repairing local radiation injury in mice.

[0033] Figure 6 The angiogenesis diagram after repairing local radiation injury in mice using Example 3 of the present invention.

[0034] Figure 7 The cumulative release amount diagram of Example 3 of the present invention and Comparative Example 1.

[0035] Figure 8 The cytotoxicity characterization results of Example 3 of the present invention and astaxanthin. Detailed implementation manners

[0036] In order to enable those skilled in the art of the present technology to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and the best embodiments.

[0037] Example 1: Preparation method of skin injury repair gel: Weigh 0.1 g of sodium alginate (molecular weight 10 kDa - 500 kDa, the same below) powder, and swell it overnight with 10 ml of deionized water to obtain a uniform and transparent sodium alginate solution; at the same time, weigh 0.1 g of hyaluronic acid (molecular weight 2 kDa - 500 kDa, the same below) powder, and swell it overnight in 10 ml of deionized water to obtain a hyaluronic acid solution with a concentration of 1%. Subsequently, add astaxanthin (7.5 mg) to 0.5 ml of the hyaluronic acid solution, stir at high speed until uniform, and after centrifugation, add the astaxanthin-hyaluronic acid solution to 1 ml of the sodium alginate solution, stir at high speed to form a uniform solution, and let it stand at room temperature to remove bubbles for 2 h. Finally, slowly add 10% calcium chloride solution (0.5 ml) along the edge of the mold to the mixed solution, and crosslink for 4 h to obtain a skin injury repair gel containing free radical removal factors. The results show that adding divalent metal ions can make the obtained skin injury repair gel have the due strength, as Figure 1 shown in a of

[0038] Example 2: Preparation method of skin injury repair gel: Weigh 0.1 g of sodium alginate powder, and swell it overnight with 10 ml of deionized water to obtain a uniform and transparent sodium alginate solution; at the same time, weigh 0.1 g of hyaluronic acid powder, and swell it overnight in 10 ml of deionized water to obtain a hyaluronic acid solution with a concentration of 1%. Add resveratrol (10 mg) to 0.5 ml of hyaluronic acid solution, stir at high speed until uniform, and after centrifugation, add the resveratrol-hyaluronic acid solution to 1 ml of sodium alginate solution, and stir at high speed to form a uniform mixed solution. Finally, add a 1% (by mass) quaternized chitosan solution (100 μl) to the mixed solution of sodium alginate solution and hyaluronic acid solution, and achieve molecular cross-linking through electrostatic interaction to obtain a skin injury repair gel containing free radical scavenging factors and antibacterial functions. The results show that although adding only the quaternized chitosan solution can also cross-link sodium alginate, and because it contains chitosan, the product has both antibacterial functions, but its cross-linking strength is insufficient and it is not suitable for application as a gel or dressing. Figure 1 shown in b.

[0039] Example 3: Preparation method of skin injury repair dressing: Weigh 0.1 g of sodium alginate powder, and swell it overnight with 10 ml of deionized water to obtain a uniform and transparent sodium alginate solution; at the same time, weigh 0.1 g of hyaluronic acid powder, and swell it overnight in 5 ml of deionized water to obtain a hyaluronic acid solution with a concentration of 2%. Add ferulic acid (10 mg), a free radical scavenging molecule, to 0.5 ml of hyaluronic acid solution, stir at high speed until uniform, and then add the ferulic acid-hyaluronic acid solution to 1 ml of sodium alginate solution, and stir at high speed to form a uniform solution. Finally, add a 1% (by mass) quaternized chitosan solution (100 μl) and a 10% calcium chloride solution (0.5 ml) to the mixed solution, and drop it onto a gauze of a suitable wound size for cross-linking to obtain a skin injury repair dressing with high strength of free radical scavenging factors and antibacterial activity. The results show that after using the quaternized chitosan solution as a cross-linking agent and using divalent ions for auxiliary cross-linking, the obtained dressing can not only remove free radicals, has antibacterial properties, but also has a strength suitable for dressing or gel application. Figure 1 shown in c.

[0040] Comparative Example 1: Preparation method of skin injury repair dressing: Weigh 0.1 g of sodium alginate powder, and swell it overnight with 10 ml of deionized water to obtain a uniform and transparent sodium alginate solution; add astaxanthin (10 mg), a free radical scavenging molecule, to 0.5 ml of sodium alginate solution, stir at high speed until uniform, drop it onto a gauze of a suitable wound size, and add a 1% (by mass) quaternized chitosan solution (100 μl) and calcium chloride solution (0.5 ml) for cross-linking to obtain a skin injury repair dressing without hyaluronic acid and with free radical scavenging factors and antibacterial activity.

[0041] Test: 1. Calculation of the water content of the skin injury repair patch: First, prepare the skin injury repair patches in Examples 1-3 above. Place the prepared gel in 0.9% physiological saline to remove unreacted small molecules. Then blot the surface moisture and weigh it to obtain the wet weight of the gel as M_wet. Figure 1 This is the morphology diagram of the skin injury repair gel patches with different sizes and strengths prepared in Examples 1-3 of the present invention. Subsequently, place the gel in an oven at 60 °C and dry it for 3 days, and weigh the dried gel as M_dry. Repeat five times, and calculate the average water content according to formula (1). The results are as Figure 2 shown. The water content of the prepared gels is all above 98%. Formula (1) Water content (%) = (M_wet - M_dry) / M_wet × 100%.

[0042] 2. Evaluation of the biocompatibility of the skin injury repair patch: The biocompatibility of the skin injury gel dressing is mainly evaluated by the cytotoxicity test of the gel patch on cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is the most commonly used method for evaluating cytotoxicity tests. First, prepare a skin injury gel containing free radical scavenging molecules and sterilize it by ultraviolet irradiation overnight. Subsequently, soak the prepared gel in DMEM complete medium containing FBS and penicillin-streptomycin and incubate it at 37 °C for 24 h to leach out soluble impurities in the gel. Inoculate 1×105 cells / ml of mouse-derived fibroblast NIH-3T3 cells into a 96-well plate and culture them in an incubator at 37 °C and 5% CO2 for 24 h. After the cells adhere and spread, aspirate the culture medium, add the gel extract, and the fresh medium group is used as the control group. After culturing in a CO2 incubator for 2 days, add MTT reagent, incubate in the dark for 4 h, then blot the solution, add 200 μl of DMSO dissolution solution, and shake it in the dark on a shaker for 10 min to completely dissolve the precipitate. Finally, pipette 100 μl of the solution into a new 96-well plate and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of the solution at 570 nm. The cytotoxicity of the hydrogel sample on cells is calculated by formula 2. The sample and the control group are set with 5 replicates to ensure that the data is statistically significant. Figure 3 This is the biocompatibility characterization result of the skin injury repair gel patch. As Figure 3 can be seen, the prepared patch samples basically have no cytotoxicity, and the cell survival rate is greater than 92%. Formula 2: Cytotoxicity (%) = (sample absorbance - blank absorbance) / (control absorbance - blank absorbance) × 100%.

[0043] 3. Evaluation of the free radical scavenging effect of the skin injury repair patch: The antioxidant effect of the radiation injury repair patch was mainly evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. First, according to Example 2, the skin injury repair gel was prepared. The patch sample (5 mm diameter gel sheet) was placed in a 0.1 mM DPPH ethanol solution, and the time for the solution to change from purple to yellow was observed. At the same time, the absorbance of the solution was measured at 30 min, 1 h, and 2 h, and the free radical scavenging rate of the gel material was calculated. Figure 4 This is the characterization result of the free radical scavenging effect of the skin injury repair gel patch prepared in Examples 1-3 of the present invention. Figure 4 As can be seen from a in the figure, at 2 h, the scavenging rate of the gel was similar to that of the pure free radical scavenging factor, and both changed from purple to yellow. Figure 4 b in the figure is the free radical scavenging rate of the gel material calculated from the absorbance at 1 h. The results show that the scavenging rate of the gel reached more than 80% at 1 h.

[0044] 3. Repair of the local radiation skin injury model in mice: A local radiation injury model in mice was constructed. After the mice were anesthetized, the skin of their hind limbs was exposed to 40 Gy of X-rays at a dose rate of 2 Gy / min for 20 minutes (Biological X-ray irradiator, Rad Source, RS 2000). After irradiation, the irradiated parts of the mice were observed every day and raised in a good environment for 10 days. When phenomena such as hair loss and swelling occurred in the irradiated parts of the mice's hind limbs, the prepared circular hydrogel patch (8 mm in diameter) was fixed on the damaged part, and a commercial wound dressing ( ) was used as a treatment control. The hydrogel dressing was changed every two days. On the 0th, 7th, and 14th days after treatment, the wound healing situation was observed and photographed. The results Figure 5 , 6 show that in the control group, ulceration and bleeding occurred at the damaged part after 7 days, while in the damaged part treated with the gel patch, there was no swelling subsidence and hair regeneration occurred. After histological section staining of the treated skin, it was found by CD31 staining that there were no new blood vessels in the skin tissue of the control group, while a large number of new blood vessels were generated in the histological section staining of the mice treated with the gel patch after 14 days.

[0045] 5. Cumulative release amount of the free radical scavenging factor before and after being coated with hyaluronic acid (HA): The skin injury gels of Example 3 and Comparative Example 1 were prepared. The patch sample (5 mm diameter gel sheet) was placed in 5 ml of PBS, placed in a 37 °C water bath shaker, the solution was taken out at the specified time, and an equal amount of fresh PBS was added. Finally, the free radical scavenging factor in the solution was quantitatively analyzed by high performance liquid chromatography. Figure 7 This is the cumulative release amount diagram of Example 3 and Comparative Example 1 of the present invention.Figure 7 It can be seen that after being coated with HA, the free radical scavenging factor improves its hydrophobic property and promotes its release rate in the gel.

[0046] 6. Cytotoxicity test of free radical scavenging factor before and after being coated with hyaluronic acid (HA): The same method as in Test 2 was used to conduct a cytotoxicity test on the hyaluronic acid-astaxanthin in Example 1 and an equal amount of pure astaxanthin molecules. From Figure 8 it can be seen that the cytotoxicity of the free radical scavenging molecule significantly decreases after being coated with hyaluronic acid.

[0047] As can be seen from the above Examples 1-3 and the comparative examples, the skin injury repair gel has good mechanical strength and biocompatibility after crosslinking. The introduction of hyaluronic acid not only compensates for the release performance of the hydrophobic free radical scavenging molecule in the skin injury repair gel, but also enhances the functional defects of the alginate gel dressing in terms of free radical scavenging and antibacterial properties. It can effectively inhibit the intracellular free radical level and promote angiogenesis, thereby promoting skin injury repair.

[0048] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a skin injury repair patch, characterized in that, It includes the following steps: Weigh 0.1 g of sodium alginate powder, and swell it overnight with 10 ml of deionized water to obtain a uniform and transparent sodium alginate solution; at the same time, weigh 0.1 g of hyaluronic acid powder, and swell it overnight in 5 ml of deionized water to obtain a hyaluronic acid solution with a concentration of 2%; add 10 mg of the free radical scavenging molecule astaxanthin to 0.5 ml of the hyaluronic acid solution, and stir it at high speed until uniform. Subsequently, add the astaxanthin-hyaluronic acid solution to 1 ml of the sodium alginate solution and stir at high speed to form a uniform solution. Finally, add 100 μl of a 1% quaternized chitosan solution and 0.5 ml of a 10% calcium chloride solution to the mixed solution, drop it onto a gauze with an appropriate wound size, and crosslink it to obtain a skin injury repair patch with a free radical removal factor and high antibacterial activity.

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