Recombinant human XVII type collagen / silibinin nanoemulsion as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicine, in particular to a recombinant human XVII type collagen / silibinin nanoemulsion and a preparation method and application thereof, and the nanoemulsion is prepared from the following components in percentage by weight: 0.2%-1.5% of recombinant human XVII type collagen, 0.5% of silibinin, 1%-10% of lecithin, 1%-10% of glycerol, 3%-15% of Tween 80, 2%-8% of PEG400 and the balance of pure water. The prepared nano-emulsion is uniform in size and high in stability, irritation is effectively reduced, skin light damage can be remarkably repaired, the synergistic effect of oxidation resistance and barrier repair is achieved, UVB-induced oxidative stress, inflammatory response and skin structure damage are inhibited in multiple ways, and the nano-emulsion has a good prospect in application of skin UVB light damage repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological agents, and in particular to a preparation method and application of recombinant human collagen type XVII / shieldin nanoemulsion. BACKGROUND

[0002] Long-term exposure of the skin to UVB radiation can cause oxidative stress, inflammatory response and tissue damage, resulting in dry skin, wrinkles, pigmentation and even photoaging, which seriously affects the health of the skin.

[0003] Shieldin is extracted from the seeds and fruits of Silybum marianum (milk thistle) and is a natural flavonoid with excellent antioxidant and anti-inflammatory activities. It has a potential protective effect on UVB-induced skin photodamage, but has defects such as poor water solubility, low transdermal absorption efficiency and insufficient bioavailability, which limit its clinical application. In the prior art, to improve the delivery efficiency of shieldin, surfactant solubilization or ordinary emulsion preparation technology is often used, but there are problems such as poor stability, limited transdermal enhancement effect and lack of synergistic effect of skin barrier repair.

[0004] Recombinant human collagen type XVII (COL17 / BP180 / BPAG2) is a transmembrane protein that is a structural component of hemidesmosomes (HD) and a key structural protein in the epidermal-dermal junction of the skin. It can promote skin barrier repair and enhance the skin delivery efficiency of hydrophobic drugs. A previous study emphasized the involvement of collagen type XVII in skin aging and wound healing and elaborated its role in healthy skin.

[0005] However, there is no related technical report on the preparation of emulsion by combining shieldin and collagen type XVII to achieve synergistic photodamage repair. Therefore, it is of great significance to develop a complex preparation with high stability, high transdermal absorption efficiency and synergistic antioxidant and anti-inflammatory effects of shieldin and skin barrier repair effects of rhCOL17 to meet the clinical needs of UVB photodamage repair. SUMMARY

[0006] To solve the above problems, the present application provides a recombinant human collagen type XVII / shieldin nanoemulsion and a preparation method and application thereof. The recombinant human collagen type XVII is non-toxic and has low irritation, and is combined with the traditional surfactant and shieldin. The recombinant human collagen type XVII used in the present application is rhCOL17, and the prepared recombinant human collagen type XVII / shieldin nanoemulsion is uniform, stable and has UVB photodamage repair effect, and has good application prospect.

[0007] To achieve the above purpose, the present application adopts the following technical solutions: The first aspect of the present application provides a recombinant human collagen type XVII / shuganling nanolipid, wherein the recombinant human collagen type XVII and shuganling are main active ingredients, and the recombinant human collagen type XVII / shuganling nanolipid is composed of 0.2% -1.5% of the recombinant human collagen type XVII, 0.5% of shuganling, 1% - 10% of lecithin, 1% - 10% of glycerol, 3%- 15% of Tween 80, 2% - 8% of PEG400, and the balance of pure water.

[0008] Further, the content of the recombinant human collagen type XVII is 1.5% by weight percentage.

[0009] Further, the content of the lecithin is 10% by weight percentage.

[0010] Further, the content of the glycerol is 2% by weight percentage.

[0011] Further, the content of the Tween 80 is 12% by weight percentage.

[0012] Further, the content of the PEG400 is 4% by weight percentage.

[0013] Further, the recombinant human collagen type XVII / shuganling nanolipid is composed of 1.5% of the recombinant human collagen type XVII, 0.5% of shuganling, 10% of lecithin, 2% of glycerol, 12% of Tween 80, 4% of PEG400, and 70% of pure water by weight percentage.

[0014] The second aspect of the present application provides a preparation method of the above-mentioned recombinant human collagen type XVII / shuganling nanolipid, and the preparation method comprises the following steps: (1) dissolving shuganling in lecithin, stirring at 20℃ - 60℃ to obtain solution A; (2) dissolving glycerol, Tween 80 and PEG400 in part of pure water, stirring uniformly to obtain solution B; (3) dissolving the recombinant human collagen type XVII in the remaining pure water to obtain collagen solution C; (4) adding solution A to solution B, stirring uniformly to form a preliminary emulsion; (5) slowly adding the collagen solution C obtained in step (3) to the preliminary emulsion, after mixing, using a high-speed shearing emulsifier for high-speed shearing treatment to obtain the recombinant human collagen type XVII / shuganling nanolipid.

[0015] Further, in step (5) of the above-mentioned preparation method, the rotation speed of the high-speed shearing treatment is 8000r / minute, and the treatment time is 2-6 minutes, preferably 6 minutes.

[0016] The third aspect of the present application provides an anti-oxidation and anti-aging pharmaceutical composition, wherein the effective component comprises the recombinant human collagen type XVII / silymarin nanoemulsion.

[0017] The fourth aspect of the present application provides the use of the recombinant human collagen type XVII / silymarin nanoemulsion in the preparation of a medicine for repairing skin photo-damage, and the skin photo-damage is UVB-induced photo-damage, which is used to solve the clinical demand for repairing UVB photo-damage.

[0018] In some embodiments of the present application, the photo-damage repairing effect is verified, and compared with the silymarin aqueous solution, the anti-UVB-induced photo-damage repairing activity can be exerted at a lower concentration, and the preparation of the recombinant human collagen type XVII (rhCOL17) / silymarin nanoemulsion can be used in the field of UVB skin photo-damage repair.

[0019] The present application has the following beneficial effects compared with the prior art: 1. The present application utilizes the skin barrier repairing effect and transdermal enhancement effect of rhCOL17 to solve the problems of poor water solubility and low delivery efficiency of silymarin, and the prepared recombinant human collagen type XVII (rhCOL17) / silymarin nanoemulsion has uniform size and strong stability, can significantly repair skin photo-damage, realizes the synergistic effect of "anti-oxidation (silymarin) + barrier repair (rhCOL17)", and multi-pathway inhibits UVB-induced oxidative stress, inflammatory response and skin structure damage.

[0020] 2. One of the raw materials of the present application, recombinant human collagen type XVII, has good biological activity and degradability, and compared with traditional surfactants, the recombinant human collagen type XVII as a surfactant can effectively reduce the irritation. At the same time, the prepared nanoemulsion can significantly improve the anti-inflammatory and anti-oxidation performance of silymarin nanoemulsion induced by UVB, so that the prepared nanoemulsion has good prospects in the application of UVB photo-damage wound. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the appearance diagram of the nanoemulsion before and after adding the recombinant human collagen type XVII; Figure 2 is the influence of different concentrations of recombinant human collagen type XVII on the particle size and dispersity of the nanoemulsion; Figure 3 is the influence of different concentrations of lecithin on the particle size and dispersity of the nanoemulsion; Figure 4 is the influence of different concentrations of glycerol on the particle size and dispersity of the nanoemulsion; Figure 5 is the influence of different concentrations of Tween 80 on the particle size and dispersity of the nanoemulsion; Figure 6 Effect of different PEG400 concentrations on the particle size and dispersity of nanoemulsion; Figure 7 Effect of different temperatures on the particle size and dispersity of nanoemulsion; Figure 8 Effect of different shearing time on the particle size and dispersity of nanoemulsion; Figure 9 Transmission electron microscopy (TEM) characterization of recombinant human collagen type XVII (rhCOL17) / silibinin nanoemulsion; Figure 10 In vitro release curve of recombinant human collagen type XVII (rhCOL17) / silibinin nanoemulsion; Figure 11 Diffusion cell transdermal experiment of recombinant human collagen type XVII (rhCOL17) / silibinin nanoemulsion; Figure 12 Effect of different concentrations of recombinant human collagen type XVII (rhCOL17) / silibinin nanoemulsion solution on the survival rate of HaCaT cells; Figure 13 Effect of each component of recombinant human collagen type XVII (rhCOL17) / silibinin nanoemulsion on the repair of UVB-induced photodamage of HaCaT cells; In the corresponding drawings, the recombinant human collagen type XVII group is abbreviated as COL17 group, the silibinin group is abbreviated as SLB group, and the recombinant human collagen type XVII (rhCOL17) / silibinin nanoemulsion group is abbreviated as SLB@COL17 NE group. DETAILED DESCRIPTION

[0022] The technical solutions of the present application are further explained below in combination with examples. Those skilled in the art can implement the present application by using other various specific embodiments according to the content disclosed in the present application, or any simple changes or modifications made by using the design structure and ideas of the present application, all fall within the protection scope of the present application. It should be noted that, in the case of no conflict, the examples in the present application and the features in the examples can be combined with each other.

[0023] In the following examples, recombinant collagen type XVII (rhCOL17) is provided by Jiangsu Chuangjian Medical Technology Co., Ltd., silibinin, lecithin, glycerol, RPMI 1640 medium, FBS, Tween 80, and PEG-400 are purchased from Shanghai Titan Technology Co., Ltd., and UVB ultraviolet light therapy instrument (SH2B) is purchased from Shanghai Sigma High-tech Co., Ltd.

[0024] Example 1 Sample preparation: 0.5% of Silybin was weighed in 10% of lecithin, and stirred uniformly in a water bath at 60°C; 2% of glycerol, 12% of Tween 80, and 4% of PEG400 were dissolved in an appropriate amount of water and stirred uniformly; 1.5% of rhCOL17 was dissolved in an appropriate amount of water for later use; the lecithin solution containing 0.5% of Silybin was slowly added to the solution of 2% of glycerol, 12% of Tween 80, 4% of PEG400 and an appropriate amount of water, and stirred uniformly to form a preliminary emulsion, which was then slowly added with the rhCOL17 aqueous solution after being placed at room temperature. The preliminary emulsion was subjected to high-speed shearing treatment for 6 minutes at a setting of 8000r / minute using a high-speed shearing emulsifier, to obtain the rhCOL17 / Silybin nanoemulsion. The high-speed shearing emulsification was performed before and after the addition of the rhCOL17 aqueous solution, respectively, and the finished products were observed as shown in Figure 1 , and Figure 1 It can be seen that after the addition of the rhCOL17 aqueous solution, the nanoemulsion is more soluble and uniformly dispersed compared with the group without rhCOL17, indicating that the addition of rhCOL17 reduces the particle size.

[0025] Example 2 Effect of different concentrations of rhCOL17, lecithin, glycerol, Tween 80 and shearing time on the nanoemulsion The nanoemulsion was prepared using 0.2%, 0.5%, 0.8%, 1%, 1.5% of rhCOL17 and 0.5% of Silybin, 10% of lecithin, 2% of glycerol, 12% of Tween 80, and 4% of PEG400, respectively, and the preparation steps were the same as in Example 1. The Malvern particle size analyzer was used for analysis and determination, as shown in Figure 2 , the nanoemulsion prepared with different concentrations of rhCOL17 has a great effect on the particle size and dispersity. When the concentration of rhCOL17 is 1.5%, the particle size and dispersity of the nanoemulsion are the lowest.

[0026] The nanoemulsion was prepared using 1%, 2%, 3%, 5%, 10% of lecithin and 1.5% of rhCOL17, 0.5% of Silybin, 2% of glycerol, 12% of Tween 80, and 4% of PEG400, respectively, and the preparation steps were the same as in Example 1. The Malvern particle size analyzer was used for analysis and determination, as shown in Figure 3 , when the concentration of lecithin is 10%, the particle size and dispersity of the nanoemulsion are the lowest.

[0027] Glycerin with concentration of 1%, 2%, 3%, 5%, 10% and recombinant human collagen type XVII with concentration of 1.5%, Silybin with concentration of 0.5%, lecithin with concentration of 10%, Tween 80 with concentration of 12%, PEG400 with concentration of 4% were used to prepare nanoemulsion, and the preparation steps were the same as those in Example 1. Malvern particle size analyzer was used for analysis and determination, as shown in Table 1. Figure 4 As shown in Table 1, when the concentration of glycerin was 2%, the particle size and dispersity of the nanoemulsion were the lowest.

[0028] Tween 80 with concentration of 3%, 6%, 9%, 10%, 12%, 15% and recombinant human collagen type XVII with concentration of 1.5%, Silybin with concentration of 0.5%, lecithin with concentration of 10%, glycerin with concentration of 2%, PEG400 with concentration of 4% were used to prepare nanoemulsion, and the preparation steps were the same as those in Example 1. Malvern particle size analyzer was used for analysis and determination, as shown in Table 2. Figure 5 As shown in Table 2, when the concentration of Tween 80 was 12%, the particle size and dispersity of the nanoemulsion were the lowest.

[0029] PEG400 with concentration of 2%, 4%, 6%, 8% and recombinant human collagen type XVII with concentration of 1.5%, Silybin with concentration of 0.5%, lecithin with concentration of 10%, glycerin with concentration of 2%, Tween 80 with concentration of 12% were used to prepare nanoemulsion, and the preparation steps were the same as those in Example 1. Malvern particle size analyzer was used for analysis and determination, as shown in Table 3. Figure 6 As shown in Table 3, when the concentration of PEG400 was 4%, the particle size and dispersity of the nanoemulsion were the lowest.

[0030] Recombinant human collagen type XVII with concentration of 1.5% and Silybin with concentration of 0.5%, lecithin with concentration of 10%, glycerin with concentration of 2%, Tween 80 with concentration of 12%, PEG400 with concentration of 4% were used to prepare nanoemulsion at temperature of 20℃, 40℃, 60℃, 80℃, and the preparation steps were the same as those in Example 1. Malvern particle size analyzer was used for analysis and determination, as shown in Table 4. Figure 7 As shown in Table 4, when the temperature was 60℃, the particle size and dispersity of the nanoemulsion were the lowest.

[0031] Recombinant human collagen type XVII with concentration of 1.5% and Silybin with concentration of 0.5%, lecithin with concentration of 10%, glycerin with concentration of 2%, Tween 80 with concentration of 12%, PEG400 with concentration of 4% were used to prepare nanoemulsion at shearing time of 2, 4, 6 minutes, and the preparation steps were the same as those in Example 1. Malvern particle size analyzer was used for analysis and determination, as shown in Table 5. Figure 8 As shown in Table 5, when the shearing time was 6 minutes, the particle size and dispersity of the nanoemulsion were the lowest.

[0032] Example 3 TEM test was performed to characterize the morphology, particle size and dispersibility of the nanoemulsion sample prepared in Example 1 according to the following steps. Sample pretreatment: the prepared recombinant human collagen type XVII (rhCOL17) / silymarin nanoemulsion sample was placed in a centrifuge tube, a 200-mesh carbon support film copper mesh was used, and the copper mesh was clamped with a dust-free tweezers. 20 μL of the nanoemulsion sample was slowly added to the surface of the copper mesh, and the sample was allowed to stand at room temperature for 3-5 min to allow the sample to fully adsorb on the surface of the support film. Drying treatment: the treated copper mesh was placed in a vacuum dryer and dried at room temperature for 8-12 h to ensure complete drying of the sample and avoid residual moisture affecting the TEM observation results. TEM test: a transmission electron microscope (model: HT-7800) was used to take TEM images. The test results showed that the TEM characterization of the nanoemulsion prepared in the application was as shown in Figure 9 From the figure, it can be seen that the nanoemulsion is spherical, the particle boundary is clear, the outline is complete, and there is no obvious adhesion or large agglomerates. The particle size distribution is <100 nm, and the dispersion uniformity is good. The results confirm that the morphology and dispersibility of the nanoemulsion are effectively controlled by screening the preparation ingredient ratio and shearing time, which provides structural stability guarantee for its application in the field of drug delivery.

[0033] Example 4 A 24 h in vitro release experiment was performed by dialysis bag method. A dialysis bag with a molecular weight cut-off of 3500 was pretreated, cut to an appropriate length, and boiled in water for 10 min. A certain amount of the nanoemulsion sample prepared in Example 1 of the application and free silymarin solution were loaded into the dialysis bag, and the two ends were tightly sealed to ensure no leakage. The dialysis bag was immersed in a beaker containing PBS buffer and placed in a 37°C constant temperature water bath with stirring at 100 rpm. At predetermined time points (e.g., 0.5, 1, 2, 4, 6, 8, 12, and 24 hours), 1 mL of release medium was collected, and an equal volume of fresh PBS buffer was supplemented. The silymarin concentration was determined by HPLC method, the cumulative release amount was calculated, and the release curve was plotted. As shown in Figure 10 The cumulative release amount of the composite emulsion reached 96.91% within 24 hours, showing good drug release kinetic characteristics.

[0034] Example 5 C57 mice were taken back skin, scraping off adipose tissue. By clamping the skin or membrane between the donor and receptor chamber, the stratum corneum upwards, fixed with O-ring to ensure no leakage, assembled Franz diffusion cell device. The receptor chamber is filled with receiving solution, excluding bubbles, and connected to the sampling system. The system is pre-equilibrated for 30-60 minutes to maintain constant temperature. The nanoemulsion sample complex emulsion prepared in Example 1 of the present application and free shugan-sijunzi solution were added to the donor chamber, respectively, and then sealed with a para film to prevent evaporation. At 0.5, 1, 2, 4, 6, 12 and 24 hours, 1 mL of receiving solution was collected, and each time a fresh receiving solution was added to maintain the "sink condition". The content was analyzed by HPLC, and a standard curve was established for quantification. As shown in Figure 11 The 24-hour cumulative transdermal amount of drug in the complex emulsion reached 46.01 μg compared with free shugan-sijunzi, significantly improving the skin penetration performance of the drug.

[0035] Example 6 HaCaT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% double antibiotic (penicillin-streptomycin) and maintained in 95% air / 5% CO2 in pups at 37°C. Cells in the logarithmic growth phase were used for experiments. Cell proliferation assay (MTT method): pre-counted cells were seeded in a 96-well plate and cultured for 24 hours. The nanoemulsion sample complex emulsion prepared in Example 1 of the present application was diluted 10x, 50x, 100x, 500x, 1000x, 1500x for administration, and then further incubated with DMEM for 24 hours. 20 μL of MTT reagent was added to each well, and incubated at 37°C for 4 hours. The culture medium was discarded, 150 μL of DMSO was added, and the plate was shaken for 10 minutes. The absorbance (OD value) was measured at 570 nm using a microplate reader as Figure 12 As shown in the figure, the complex emulsion at a concentration of 1500x significantly promoted cell proliferation, so this concentration was determined as the optimal working concentration for subsequent experiments.

[0036] Example 7 UVB-induced photoaging model establishment: The pre-counted cells were inoculated in 96-well plates and grouped: control group (Control), UVB modeling group (UVB), shikonin empty emulsion group (SLB NE), recombinant human collagen type XVII empty emulsion group (COL17 NE), recombinant human collagen type XVII (rhCOL17) / shikonin nanoemulsion group (SLB@COL17 NE) and cultured for 24 hours. The culture medium was discarded, and 20 μL PBS was added to cover the bottom of each well. Except for the modeling wells, all wells were covered with tin paper. The UVB phototherapy instrument was placed 15 centimeters above the console for irradiation. After irradiation, the PBS was discarded, and the culture medium was replaced with serum-free DMEM or other treatment medium for further culture. The UVB irradiation dose of 1000 mJ / cm² can stably induce a cell photo-damage model, so it is selected as the modeling parameter for subsequent experiments. Based on this, the post-damage administration experiment is as shown in Figure 13 The recombinant human collagen type XVII (rhCOL17) / shikonin nanoemulsion plays a certain repair role on UVB-induced cell photo-damage, providing preliminary experimental evidence for its application in photo-damage treatment.

[0037] The above-described embodiments only express several embodiments of the present application, which are described in detail and in detail, but cannot be understood as limiting the scope of the patent application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims.

Claims

1. A recombinant human type XVII collagen / silymarin nanoemulsion, characterized in that, By weight percentage, it consists of the following components: 0.2% - 1.5% recombinant human type XVII collagen, 0.5% silymarin, 1% - 10% lecithin, 1% - 10% glycerol, 3% - 15% Tween 80, 2% - 8% PEG400, and the balance being purified water.

2. The recombinant human type XVII collagen / silymarin nanoemulsion according to claim 1, characterized in that, The content of the recombinant human type XVII collagen is 1.5% by weight.

3. The recombinant human type XVII collagen / silymarin nanoemulsion according to claim 1, characterized in that, The lecithin content is 10% by weight.

4. The recombinant human type XVII collagen / silymarin nanoemulsion according to claim 1, characterized in that, The glycerol content is 2% by weight.

5. The recombinant human type XVII collagen / silymarin nanoemulsion according to claim 1, characterized in that, The content of Tween 80 is 12% by weight.

6. The recombinant human type XVII collagen / silymarin nanoemulsion according to claim 1, characterized in that, By weight percentage, it consists of the following ingredients: 1.5% recombinant human type XVII collagen, 0.5% silymarin, 10% lecithin, 2% glycerin, 12% Tween 80, 4% PEG400, and 70% purified water.

7. The method for preparing recombinant human type XVII collagen / silymarin nanoemulsion according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Dissolve silymarin in lecithin and stir at 20℃ - 60℃ to obtain solution A; (2) Dissolve glycerol, Tween 80 and PEG400 in some pure water and stir well to obtain solution B; (3) Dissolve recombinant human type XVII collagen in the remaining pure water to obtain collagen solution C; (4) Add solution A to solution B and stir to form a primary emulsion; (5) Slowly add the collagen solution C obtained in step (3) to the colostrum, mix, and then perform high-speed shearing treatment using a high-speed shear emulsifier to obtain the recombinant human type XVII collagen / silymarin nanoemulsion.

8. The preparation method according to claim 7, characterized in that, In step (5), the rotation speed of the high-speed shearing process is 8000 r / min, and the processing time is 2-6 minutes, preferably 6 minutes.

9. An antioxidant and anti-aging pharmaceutical composition, characterized in that, Its active ingredient comprises the recombinant human type XVII collagen / silymarin nanoemulsion as described in any one of claims 1-6.

10. The use of the recombinant human type XVII collagen / silymarin nanoemulsion as described in any one of claims 1-6 in the preparation of a medicament for repairing photodamage to the skin, characterized in that, The skin photodamage mentioned is photodamage caused by UVB.

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