Preparation method and application of recombinant XVII type collagen and adenosine eutectic

Abstract

The invention discloses a preparation method and application of a recombinant XVII type collagen and adenosine eutectic substance. According to the invention, adenosine and recombinant XVII type collagen are combined through a non-covalent bond to form a novel supramolecular eutectic substance. The solubility and skin permeability of the eutectic substance are improved through the synergistic effect of the two substances, and the performance of fixing hair and promoting hair papilla cell proliferation is also enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of a recombinant type XVII collagen and adenosine cocrystal and an application thereof. Background Art

[0002] Adenosine is a commonly used active ingredient in cosmetics. Adenosine has multiple activities, such as analgesia, anti-inflammation, and promotion of wound healing. Adenosine is also widely used in cosmetics and personal care products. It can promote the production of collagen in human dermal fibroblasts, improve skin elasticity, reduce wrinkles around the eyes and between the eyebrows, and has the effects of moisturizing, repairing, and stimulating hair growth. Despite these positive effects of adenosine, it also faces some challenges in practical application. For example, the solubility of adenosine in water is relatively low (less than 1% at room temperature), which further limits its effect in the product.

[0003] Recombinant type XVII collagen is a transmembrane protein with the highest expression in the skin, containing 1497 amino acids. Collagen 17 plays an important role in maintaining the structure and function of the skin, especially in the basement membrane area between the epidermis and the dermis, where it helps the epidermal cells to connect tightly to the basement membrane, thereby maintaining the integrity and elasticity of the skin. It also helps prevent premature hair loss and aging of hair follicles, and has potential effects on hair care and anti-hair loss. In addition, recombinant type XVII collagen performs poorly in terms of skin permeability, which also limits its effect in products. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides a method for preparing a co-crystal of recombinant type XVII collagen and adenosine, characterized in that it comprises the following steps:

[0006] The recombinant type XVII collagen and adenosine are mixed and added into a solvent, stirred at room temperature, and dried at 40-50° C. to obtain a solid powdery eutectic.

[0007] As a preferred embodiment of the method for preparing the co-crystal of recombinant type XVII collagen and adenosine of the present invention, the mass ratio of the recombinant type XVII collagen to adenosine is 1:4-6.

[0008] As a preferred embodiment of the method for preparing the co-crystal of recombinant type XVII collagen and adenosine of the present invention: the solvent is water, and the mass ratio of the total mass of recombinant type XVII collagen and adenosine to water is 1:1-20.

[0009] As a preferred embodiment of the method for preparing the co-crystal of recombinant type XVII collagen and adenosine of the present invention: the solvent is water, and the mass ratio of the total mass of recombinant type XVII collagen and adenosine to water is 1:3-10.

[0010] As a preferred embodiment of the method for preparing the recombinant type XVII collagen and adenosine co-crystal of the present invention, the stirring time is 20 to 40 minutes.

[0011] Beneficial effects of the present invention: The present invention uses adenosine and recombinant type XVII collagen to form a new supramolecular eutectic material by non-covalent bonding. The synergistic effect of the two substances improves the solubility and skin permeability of the eutectic material, and also has an enhanced effect on hair strengthening and promoting hair papilla cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0013] Figure 1 This is the XRD spectrum of the co-crystal of recombinant type XVII collagen and adenosine.

[0014] Figure 2 This is the infrared spectrum of the co-crystal of recombinant type XVII collagen and adenosine.

[0015] Figure 3 This is a graph showing the penetration of recombinant type XVII collagen and adenosine co-crystal into pig skin at different times.

[0016] Figure 4 This is a graph of the proliferation rate of dermal papilla cells.

[0017] Figure 5 This is a graph showing the content of vascular endothelial growth factor in hair papilla cells. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0019] Embodiment 1:

[0020] Preparation method of recombinant type XVII collagen and adenosine cocrystal: recombinant type XVII collagen (CAS: 9064-67-9, humanized recombinant collagen) powder and adenosine (CAS: 58-61-7) powder are mixed in a mass ratio of 1:5 and added into deionized water, with a solid-liquid ratio of 1:6, and stirred at 300 rpm for 30 minutes at room temperature. After stirring, the water is dried at 40°C to precipitate solid powdery cocrystals.

[0021] Preparation method of control sample: recombinant type XVII collagen (CAS: 9064-67-9) powder and adenosine (CAS: 58-61-7) powder were mixed at a mass ratio of 1:5, and ground with a pestle for 10 minutes to obtain a physical mixing group powder as a control.

[0022] The solid powder obtained after drying was subjected to the following experimental tests:

[0023] (1) The powder was subjected to an X-ray diffraction test to explore its crystalline state. A nickel-filtered Cu (Kα) radiation with a wavelength of 0.154 nm and a 2θ angle of 5 to 60° was used to continuously scan and obtain the diffraction pattern.

[0024] (2) Structural analysis of the substance is performed using Fourier total reflection infrared spectroscopy (FTIR).

[0025] (3) Solubility test:

[0026] At room temperature, add 10 mL of deionized water to a 25 mL conical flask with a glass stopper, and then add an excess of the prepared co-crystals to the conical flask. Stir and vibrate for 6 hours to ensure that solid-liquid equilibrium is reached. After standing for 24 hours, use a preheated pipette to take approximately 1 g of saturated supernatant from each round-bottom flask, then place it in a pre-weighed beaker, and then immediately measure the total weight of the filtrate in the beaker. After that, place the beaker in a closed dry environment and leave it naturally until the solvent is completely dry, and then weigh the beaker again. Each experiment was repeated 3 times, and the average value was taken to calculate the solubility. The calculation formula is as follows:

[0027]

[0028] (4) Skin penetration test:

[0029] Using the improved Franz diffusion cell method, the pig skin was fixed between the supply cell and the receiving cell, with the stratum corneum facing the supply cell. The receiving solution was a phosphate buffer solution with a pH of 6.8. The receiving cell was kept in a constant temperature water bath at (37±0.2)°C and magnetically stirred at a speed of 300r / min. The eutectic was prepared into a 10mg / mL aqueous solution, 1mL of the eutectic solution was added to the supply cell, and 0.75mL of the receiving solution was taken at 1, 2, 4, and 8 hours, respectively. At the same time, the receiving cell was supplemented with an equal volume of blank receiving solution at the same temperature. After the removed receiving solution was filtered, its content was determined, and the cumulative permeation amount Q at each time point was calculated according to the following formula n (μg / cm 2 ):

[0030]

[0031] In the formula, C n is the mass concentration of the eutectic in the receiving solution at the nth sampling point, V is the volume of the receiving pool (7 mL), V0 is the sampling volume at each time point, C i is the mass concentration of eutectic in the receiving solution at the i-th sampling point (i≤n-1), and A is the effective diffusion cell area (2.2cm 2 ). Plot the Qt graph with the cumulative permeation amount Qn versus time t, and perform model fitting.

[0032] (5) Dermal papilla cell proliferation rate test:

[0033] Digest the hair papilla cells in the logarithmic growth phase and count them using a hemocytometer. Inoculate 1×104 cells per well in a 96-well cell culture plate. After 24 hours of culture, discard the supernatant. Add different concentrations of eutectic solutions dissolved in the culture medium and physical mixed solutions of collagen and adenosine to the sample wells, respectively. The blank control group was without sample, and the group without cells and samples was withered. Multiple replicates were set for each well. After 24 hours of culture, discard the supernatant and wash with PBS. Add 100 μL of freshly prepared MTT solution of 0.5 mg / mL (dissolved in DMEM culture medium) to each well. After incubation for 4 hours, discard the supernatant, add 100 μL of DMSO to each well to dissolve the crystals, shake at 37°C for 5 minutes, read the absorbance at 490 nm on an ELISA reader, and calculate the cell proliferation formula as follows:

[0034]

[0035] Wherein: A0 represents the absorbance value of the solvent group; A1 represents the absorbance value of the sample group; A2 represents the absorbance value of the blank control group.

[0036] (6) Determination of vascular endothelial growth factor (VEGF) in hair papilla cells:

[0037] Cell pretreatment was the same as above. After the cells were cultured for 24 hours and adhered to the wall, the complete medium was aspirated and the bottom of the plate was rinsed twice with PBS. 2 mL of freshly prepared eutectic solution of a certain concentration was added to each well. A blank group, sample groups of different mass concentrations, and a physical mixture of collagen and adenosine were set up and cultured for 48 hours. The VEGF concentration in the culture medium was determined according to the EISA kit operating instructions.

[0038] The test results of the co-crystal are as follows:

[0039] (1) XRD spectrum of recombinant type XVII collagen and adenosine co-crystal is shown in Figure 1The characteristic peaks of adenosine are 11.71°, 15.39°, 17.70°, 19.11°, 20.75°, 21.45°, 23.16°, 27.30°, 29.00° and 38.51°, and recombinant type XVII collagen has a broad peak near 21.28°. The XRD spectrum of the co-crystal of recombinant type XVII collagen and adenosine changes with new characteristic peaks at 5.53°, 7.23°, 13.53° and 16.36°, while the material of the physical mixture of recombinant type XVII collagen and adenosine does not have new characteristic peaks, which means that the method of the present invention successfully prepares the co-crystal of recombinant type XVII collagen and adenosine.

[0040] (2) Infrared images of recombinant type XVII collagen and adenosine are shown in Figure 2 The infrared images of the mixture and co-crystal of recombinant type XVII collagen and adenosine are shown in Figure 2 The -NH2 of adenosine is at 3320cm- 1 The stretching vibration of NH is at 1660cm- 1 After recombinant type XVII collagen forms a cocrystal with adenosine, the -NH2 of adenosine red-shifts to 3307cm- 1 , the stretching vibration peak of NH red-shifted to 1654 cm- 1 At the same time, the peak intensity in the co-crystal is weaker than that in the single component. The above phenomenon indicates that there are weak interactions such as hydrogen bonds between adenosine and collagen, which provides evidence for the formation of a co-crystal structure.

[0041] (3) Solubility test:

[0042]

[0043] (4) Cumulative permeation of recombinant type XVII collagen and adenosine co-crystal (Q n ) and time (t) as shown in the figure Figure 3 As shown in Figure 2, the cumulative permeation amount (Q n The permeability of the co-crystal at the four sampling times of 1, 2, 4, and 8 h was higher than that of recombinant type XVII collagen. The cumulative permeability after 8 h of dialysis increased by 52.9% compared with that of recombinant type XVII collagen, indicating that the co-crystal has a good permeability-enhancing effect.

[0044] (5) Figure 4The co-crystal has a proliferation effect on hair papilla cells. The co-crystal solution is non-toxic to cells in the concentration range of 0.1wt%-1wt%, and shows a more significant proliferation effect on hair papilla cells at 0.2% and 0.5% (p<0.01), with proliferation rates of 126.31% and 122.64%, respectively. The physical mixture of collagen and adenosine has no significant proliferation effect compared to the blank group (p>0.05), indicating that the co-crystal has a certain potential in preventing hair loss.

[0045] (6) Figure 5 The concentration of VEGF secreted by hair papilla cells. The VEGF content of the cell supernatant in the eutectic solution within the concentration range of 0.05%-1% was higher than that in the blank group, and the 0.2% and 0.5% eutectic solutions significantly increased VEGF secretion by 32.1% and 44.1% (p<0.001), respectively, which has a significant potential to promote hair growth.

[0046] The present invention uses adenosine and recombinant type XVII collagen to form a new supramolecular eutectic material by non-covalent bonding. The synergistic effect of the two substances improves the solubility and skin permeability of the eutectic material, and also has an enhanced effect on hair strengthening and promoting hair papilla cell proliferation.

[0047] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a co-crystal of recombinant type XVII collagen and adenosine, characterized in that: It consists of the following steps: The recombinant type XVII collagen and adenosine are mixed and added into a solvent, stirred at room temperature, and dried at 40-50° C. to obtain a solid powdery eutectic.

2. The method for preparing the recombinant type XVII collagen and adenosine cocrystal according to claim 1, characterized in that: The mass ratio of the recombinant type XVII collagen to adenosine is 1:4-6.

3. The method for preparing the recombinant type XVII collagen and adenosine cocrystal according to claim 1 or 2, characterized in that: The solvent is water, and the mass ratio of the total mass of the recombinant type XVII collagen and adenosine to water is 1:1-20.

4. The method for preparing the recombinant type XVII collagen and adenosine cocrystal according to claim 1 or 2, characterized in that: The stirring time is 20 to 40 minutes.

5. The method for preparing the co-crystal of recombinant type XVII collagen and adenosine according to claim 3, characterized in that: The solvent is water, and the mass ratio of the total mass of the recombinant type XVII collagen and adenosine to water is 1:3-10.

6. The co-crystal prepared by the method for preparing the co-crystal of recombinant type XVII collagen and adenosine according to claim 1.

7. The role of the co-crystal prepared by the method for preparing the co-crystal of recombinant type XVII collagen and adenosine according to claim 1 in preparing a product for promoting the proliferation of dermal papilla cells.

8. The role of the co-crystal prepared by the method for preparing the co-crystal of recombinant type XVII collagen and adenosine according to claim 1 in preparing cosmetics.

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