Acidic fibroblast growth factor transdermal material as well as preparation and application thereof

Abstract

The invention discloses an acidic fibroblast growth factor transdermal material as well as preparation and application thereof. The material is prepared from acidic fibroblast growth factors, graphene oxide and polypropylene fibers, the polypropylene fiber is laid at the bottom of the graphene oxide to obtain a graphene oxide polypropylene fiber scaffold; the acidic fibroblast growth factors F are uniformly distributed on the surface layer of the graphene oxide polypropylene fiber scaffold. The problem that in the prior art, the transdermal stability of aFGF is low is solved. Compared with the prior art, the aFGF-GO provided by the invention can reduce the pain caused by a microneedle, does not chemically react with the skin, and can reduce the damage to the skin; the preparation process of the aFGF-GO is simple, the adopted GO belongs to a biosafety material, and the prepared aFGF-GO increases the stability of the aFGF on the premise of not influencing the biological activity of the aFGF and has good transdermal efficiency.

Description

Technical Field

[0001] The present invention relates to a transdermal material, in particular to an acidic fibroblast growth factor transdermal material and its preparation and application. Background Art

[0002] Acidic fibroblast growth factor (aFGF) was isolated and purified from bovine brain by Thomas et al. in 1984. With an acidic isoelectric point (pI) of 5-7, it is the first member of the fibroblast growth factor family and is also known as FGF-1. aFGF is a normal, trace substance found in mammalian tissues, primarily distributed in organs and tissues such as the brain and kidneys. It has a mitogenic effect on a variety of cells derived from the mesoderm and neuroectoderm. The amino acid sequence of aFGF was elucidated in the 1980s. Since the 1990s, advances in genetic engineering have enabled the large-scale expression of highly active and pure aFGF in Escherichia coli, enabling its widespread clinical application and greatly expanding its scope of application.

[0003] aFGF is a kind of important mitogen, can promote cell division, proliferation and participate in the migration and differentiation of cells during embryonic development. aFGF can make ulcer be repaired quickly by promoting the proliferation of blood vessels at the bottom of ulcer. In addition, endogenous acidic fibroblast growth factor and its receptor expression level are all improved in damaged cerebral cortex, which explains that aFGF plays an important role in wound healing, protection and regeneration of nervous system. aFGF also has the effect of promoting hair growth. The effect of aFGF promoting hair growth has been reported, and its mechanism has also been further studied. However, the key of promoting hair growth is the stability and transdermal efficiency of transdermal drugs. Therefore, for improving the stability and transdermal rate of aFGF in the promotion of hair growth, problems needing urgent solution are formed.

[0004] To address these issues, existing technologies employ several approaches to increase the biostability and transdermal permeability of aFGF, such as using microneedles to deliver aFGF directly to the dermis. Other approaches include fusing aFGF with transdermal molecules such as penetrating peptides to increase its success rate. However, all of these approaches can irreversibly affect the stability and biological activity of aFGF, while also impacting the user experience. Therefore, a method that increases aFGF transdermal efficiency without compromising stability is urgently needed. Summary of the Invention

[0005] The purpose of the present invention is to provide an acidic fibroblast growth factor transdermal material and its preparation and application, which solves the problem of low transdermal stability of aFGF in the prior art.

[0006] In order to achieve the above object, the present invention provides an acidic fibroblast growth factor transdermal material, which: The invention is composed of acidic fibroblast growth factor, graphene oxide and polypropylene fiber; the polypropylene fiber is laid on the bottom of the graphene oxide (or described as: graphene oxide is doped and distributed on the polypropylene fiber) to obtain a graphene oxide polypropylene fiber scaffold; the acidic fibroblast growth factor F is evenly distributed on the surface of the graphene oxide polypropylene fiber scaffold.

[0007] The present invention provides a method for preparing the material as described above, the method comprising: (1) dissolving graphene oxide in a hexafluoroisopropanol solution to obtain a mixed solution, and soaking polypropylene fiber in the obtained mixed solution to obtain a fiber material; cooling the obtained fiber material at -20°C, and freeze-drying it in a vacuum to obtain a graphene oxide polypropylene fiber scaffold; (2) Loading a phosphate buffer solution containing acidic fibroblast growth factor into the graphene oxide nanofiber scaffold obtained in step (1), and allowing to stand at room temperature to obtain the acidic fibroblast growth factor transdermal material, which is recorded as aFGF-GO.

[0008] Preferably, in step (1), the mass ratio of the graphene oxide to the hexafluoroisopropanol solution is 0.2:98.8.

[0009] Preferably, in step (1), the mass fraction of hexafluoroisopropanol in the hexafluoroisopropanol solution is 6%.

[0010] Preferably, in step (2), the concentration of the acidic fibroblast growth factor in the phosphate buffer solution containing the acidic fibroblast growth factor is 0.05%.

[0011] Preferably, in step (2), the loading amount is 1 μg / mL.

[0012] Preferably, in step (2), the standing time is 24 hours.

[0013] Preferably, in step (2), the acidic fibroblast growth factor is separated and purified by histidine affinity chromatography and ion exchange chromatography.

[0014] Preferably, the solution A used in the purification is 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 10 mM β-mercaptoethanol, pH 8.0; and the solution B is 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 1 M imidazole, 10 mM β-mercaptoethanol, pH 8.0.

[0015] The present invention also provides a use of the material in promoting hair growth.

[0016] The acidic fibroblast growth factor transdermal material, preparation, and application of the present invention solve the problem of low transdermal stability of aFGF in the prior art and have the following advantages: 1. Compared with the existing technology, the aFGF-GO of the present invention can reduce the pain caused by microneedles and does not react chemically with the skin, thereby reducing damage to the skin.

[0017] 2. Compared with the prior art, the aFGF-GO preparation process of the present invention is simple, and the GO used is a biosafe material. The prepared aFGF-GO increases the stability of aFGF without affecting the biological activity of aFGF, and has good transdermal efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the expression and purification results of aFGF of the present invention.

[0019] Figure 2 Schematic diagram of aFGF-GO prepared in the present invention.

[0020] Figure 3 is the porosity of aFGF-GO prepared in the present invention.

[0021] Figure 4 is the swelling rate of aFGF-GO prepared in the present invention.

[0022] Figure 5 This is a scanning electron microscope image of aFGF-GO prepared in the present invention.

[0023] Figure 6 This is a diagram showing the hair growth-promoting effect of applying the aFGF-GO prepared in the present invention on the back of hairless mice for 14 days.

[0024] Figure 7 This is a graph showing the test results of the transdermal release ability of aFGF-GO prepared in the present invention. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0026] The culture media and reagents involved in the following examples are specifically as follows: The culture medium was DMEM containing 10% FBS and double antibodies.

[0027] Phosphate buffer contains 0.01 M phosphate.

[0028] Solution A is 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 10 mM β-mercaptoethanol, pH 8.0; solution B is 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 1 M imidazole, 10 mM β-mercaptoethanol, pH 8.0.

[0029] Example 1 A method for preparing an acidic fibroblast growth factor transdermal material, the method comprising: (1) Preparation of aFGF The engineered bacteria expressing aFGF were inoculated (the volume of the engineered bacteria expressing aFGF was one thousandth of the volume of the culture medium) in a fermentation medium containing Amp resistance (the volume of Amp resistance was one thousandth of the volume of the culture medium). When the optical density OD ≥ 1, IPTG (isopropylthiogalactoside) was added to induce expression for 4 hours, and then the bacteria were collected. A certain amount of lysis buffer (the mass ratio of lysis buffer to bacteria was 20:1) was added, and the bacteria were disrupted by ultrasound. The aFGF was separated and purified using histidine affinity chromatography and ion exchange chromatography.

[0030] Affinity chromatography: ①Pump cleaning: First clean the entire system with 200~300 ml of deionized water at 2.5 ml / mins.

[0031] ② Equilibration: Wash with 100% solution B at 2.5 ml / min for 8-10 column volumes, then switch to 100% solution A at 1.5 ml / min until the conductivity and UV tend to be stable (Solution A is 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 10 mM β-mercaptoethanol, pH 8.0; Solution B is 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 1 M imidazole, 10 mM β-mercaptoethanol, pH 8.0, same as below).

[0032] ③ Sample loading: Adjust the flow rate to 1 ml / min. When adding the sample, collect the effluent until the UV and conductivity values ​​are stable.

[0033] ④ Re-balance: After the sample solution is applied, replace it with 100% A solution and continue to balance for 8 to 10 column volumes (40 to 50 ml).

[0034] ⑤ Elution: After the balance solution has flowed out, add eluent (300mM imidazole) to elute the sample. When the peak appears, collect the effluent. After washing with wash solution 1, continue washing for 8 to 10 column volumes until UV and conductivity are stable.

[0035] ⑥ End: After the washing solution is finished and the baseline is completely lowered, use 50%A+50%B water to wash the pipes and columns.

[0036] Anion column chromatography: First, wash the column with 100% Solution B at 1.5 ml / min, fixing the volume to 50 ml. Then, equilibrate with 100% Solution A at 1.5 ml / min. After equilibration, apply sample at 1 ml / min. After 1-2 column volumes, collect the effluent. Wait until the peak decreases and stabilizes before stopping collection. Then, equilibrate with 100% Solution A for 8-10 column volumes. After equilibration, elute the sample with Solution B at 5% (0.1 mol), 35% (0.7 mol), and 100% (2 mol). After elution, wait for baseline equilibrium, then rinse the AB tubing and column with water (approximately 100 ml).

[0037] like Figure 1 As shown in the figure, the expression and purification results of aFGF of the present invention, wherein M is 26619 protein marker; 1 is the total protein of the bacteria before induction; 2 is the total protein of the bacteria after induction; 3 is the elution of histidine affinity chromatography with 0.2 mol imidazole; 4 is the elution of DEAE ion exchange chromatography with 0.1 mol NaCl. Figure 1 It was found that purified aFGF was obtained by the two-step purification method.

[0038] (2) Preparation of polypropylene fiber Polypropylene fibers were placed in an ozone environment at a concentration of 500 ppm and irradiated with 254 nm UV light for half an hour. Ozone induces an oxidation reaction on the polypropylene fiber surface, breaking some C–H bonds and generating free radicals. These free radicals react with oxygen to form oxidized groups, such as carboxyl (-COOH) and hydroxyl (-OH). After irradiation, the polypropylene fibers were air-dried at room temperature to ensure the stability of the surface functional groups.

[0039] (3) Preparation of aFGF-GO materials 0.48 mg of graphene oxide was dissolved in a 6% hexafluoroisopropanol solution to obtain a mixed solution (wherein the mass of graphene oxide in the mixed solution was 0.2% of the total mass of the mixed solution), and 1 g of polypropylene fiber was soaked in the obtained mixed solution to obtain a fiber material; the obtained fiber material was cooled at -20°C and then freeze-dried in a vacuum freeze dryer to obtain a graphene oxide polypropylene fiber scaffold; 1 μL / mL of acidic fibroblast growth factor was dissolved in a phosphate buffer solution to obtain a growth factor solution, and the obtained growth factor solution was loaded into the obtained graphene oxide polypropylene fiber scaffold at a concentration of 1 μg / mL, and the mixture was allowed to stand at room temperature for 24 hours to prepare a sterile aFGF-GO transdermal material.

[0040] like Figure 2 As shown in FIG, a schematic diagram of aFGF-GO prepared by the present invention. Figure 2 It can be seen that the aFGF-GO prepared in the present invention includes a hair growth promoting agent aFGF purified protein, graphene oxide (GO) skin penetration aid and polypropylene fiber support.

[0041] like Figure 3 As shown in the figure, the porosity of aFGF-GO prepared by the present invention, wherein PP is polypropylene; GO-PP is aFGF-GO, and the vertical axis is porosity. Figure 3 It can be seen that the internal space of aFGF-GO allows for drug encapsulation.

[0042] like Figure 4 As shown in the figure, the swelling rate of aFGF-GO prepared by the present invention, wherein PP is polypropylene; GO-PP is aFGF-GO, and the vertical axis is the swelling rate. Figure 3 It can be seen that aFGF-GO has a good drug encapsulation capacity.

[0043] like Figure 5 As shown in FIG, a scanning electron microscope image of aFGF-GO prepared by the present invention. Figure 5 It can be seen that aFGF-GO has a loose porous structure.

[0044] Example 2 (1) Establishment of animal model of hair loss Hair plucking was used to induce hair loss and establish an animal model of hair loss: two groups of 7-week-old female C57BL / 6 mice (4 mice in each group) were anesthetized using a small animal gas inhalation anesthesia machine; rosin and paraffin were mixed in a mass ratio of 1:1, heated and melted, and evenly applied to the backs of the mice. After the depilatory wax solidified, it was removed. The above application, solidification, and removal operations were repeated three times until the backs of the mice were smooth and hairless, resulting in two groups of mice with hair loss.

[0045] (2) Detection of the hair growth-promoting effect of aFGF-GO The specific process of the test is as follows: on the 1st, 4th, 7th and 10th days, 20μM aFGF-GO material was applied to the backs of the first group of mice with hair loss. The second group of mice was not given 20μM aFGF-GO material, and other conditions were the same as the first group (approximately 0.05g of aFGF-GO material was applied to the back of each mouse), serving as a blank control group; starting from the third day, the hair growth on the backs of each group of mice was recorded every day with a camera, and the hair growth in the administration area was photographed with a hair follicle detector. At the same time, the skin tissue in the administration area and the skin tissue at the same position in the second group were collected to obtain the collected tissue; the collected tissue was immersed in 4% paraformaldehyde and fixed for 24~48h without shaking during the period; the fixed skin was rinsed with water to remove excess paraformaldehyde. The tissue was then dehydrated with 70%, 80%, 90% and 100% ethanol in sequence, and xylene was added for transparency. The treated tissue was embedded in melted paraffin, cooled and solidified at room temperature after embedding, and the tissue was cut into 10 μm thick slices using a paraffin slicer. The slices were naturally air-dried, immersed in xylene, and dewaxed to obtain dewaxed tissue. The dewaxed tissue was fully hydrated with 100%, 90%, 80% and 70% ethanol in sequence, and then stained with hematoxylin, rinsed with deionized water, and stained with eosin to obtain skin slices to be tested for hair growth-promoting effects.

[0046] like Figure 6 As shown in the figure, the invention applied the prepared aFGF-GO to the back of hairless mice for 14 days to promote hair growth, where A is a skin section of a mouse without aFGF-GO; B is a skin section of a mouse with aFGF-GO. Figure 3 It can be seen that the aFGF-GO prepared in the present invention has the effect of changing the hair cycle and promoting hair regeneration.

[0047] (3) Detection of aFGF-GO transdermal release ability The specific detection process is as follows: the prepared skin tissue sections are heated under high pressure, removed and cooled, washed with PBS, and blocked with 30 μL of 5% BSA for 1 hour; the blocking solution is washed off, and a 1:250 dilution of anti-aFGF antibody is added and incubated at 4°C overnight. The primary antibody on the tissue is washed off, and a 1:500 dilution of the corresponding secondary antibody is added and incubated at room temperature for 1 hour (blocking is done with 30 μL of 5% BSA for 1 hour, and 20 μL of primary antibody is added to each tissue and incubated overnight at 4°C. The secondary antibody is also incubated with 20 μL of secondary antibody at room temperature for 1 hour). The sections are then air-dried to obtain air-dried tissue sections; 200 μL of DAB (diaminobenzidine) colorimetric solution is added to the air-dried tissue sections, and the sections are examined under a microscope. Staining is stopped when the tissue turns brownish-yellow after the addition of the colorimetric solution; the tissue sections are washed and dehydrated in 70%, 80%, 90%, and 100% ethanol in sequence; after the tissue is completely dehydrated, the sections are mounted with neutral resin and images are obtained by microscopic observation.

[0048] like Figure 7 As shown in the figure, the test results of the transdermal release ability of aFGF-GO prepared by the present invention are shown, wherein A is the immunofluorescence test of hair follicles given aFGF; B is the immunofluorescence test of hair follicles given aFGF-GO. Figure 4 It can be seen that the graphene oxide in the aFGF-GO prepared in the present invention has a significant effect of promoting drug transdermal penetration.

[0049] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An acidic fibroblast growth factor transdermal material, characterized in that: The material: It is composed of acidic fibroblast growth factor, graphene oxide and polypropylene fibers; The polypropylene fiber is laid on the bottom of the graphene oxide to obtain a graphene oxide polypropylene fiber scaffold; The acidic fibroblast growth factor F is evenly distributed on the surface of the graphene oxide polypropylene fiber scaffold.

2. A method for preparing the material according to claim 1, characterized in that: The method includes: (1) dissolving graphene oxide in a hexafluoroisopropanol solution to obtain a mixed solution, and soaking polypropylene fiber in the obtained mixed solution to obtain a fiber material; cooling the obtained fiber material at -20°C, and freeze-drying it in a vacuum to obtain a graphene oxide polypropylene fiber scaffold; (2) Loading a phosphate buffer solution containing acidic fibroblast growth factor into the graphene oxide nanofiber scaffold obtained in step (1), and allowing to stand at room temperature to obtain the acidic fibroblast growth factor transdermal material, which is recorded as aFGF-GO.

3. The method according to claim 2, characterized in that In step (1), the mass ratio of the graphene oxide to the hexafluoroisopropanol solution is 0.2:98.

8.

4. The method according to claim 2, characterized in that In step (1), the mass fraction of hexafluoroisopropanol in the hexafluoroisopropanol solution is 6%.

5. The method according to claim 2, characterized in that In step (2), the concentration of the acidic fibroblast growth factor in the phosphate buffer solution containing the acidic fibroblast growth factor is 0.05%.

6. The method according to claim 2, characterized in that In step (2), the loading amount is 1 μg / mL.

7. The method according to claim 2, characterized in that In step (2), the standing time is 24 hours.

8. The method according to claim 2, characterized in that In step (2), the acidic fibroblast growth factor is separated and purified by histidine affinity chromatography and ion exchange chromatography.

9. The method according to claim 8, characterized in that The solution A used in the purification was 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 10 mM β-mercaptoethanol, pH 8.0; the solution B was 20 mM Tris-HCl, 6 M urea, 0.2 M NaCl, 1 M imidazole, 10 mM β-mercaptoethanol, pH 8.

0.

10. Use of the material according to claim 1 in promoting hair growth.

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