Use of a protein nanometer selenium hydrogel in preparation of a medicine for preventing and treating androgenetic alopecia

Abstract

The application belongs to the technical field of biological medicine, and discloses a use of a protein nano selenium hydrogel in preparation of a medicine for preventing and treating androgenetic alopecia. The protein nano selenium hydrogel is used to prevent and treat androgenetic alopecia, and has stronger antioxidant activity, better drug efficacy and good safety compared with existing inorganic nano selenium. Pharmacodynamic research shows that the protein nano selenium has better efficacy in a mouse model of androgenetic alopecia than inorganic nano selenium, melanin pigmentation appears earlier, and hair follicle cells can be activated to enter a hair growth phase earlier.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the use of a protein nano-selenium hydrogel in the preparation of drugs for the prevention and treatment of androgenetic alopecia. Background Technology

[0002] Androgenetic alopecia (AGA) is a common type of progressive hair loss in clinical practice. It is an androgen-dependent genetic disorder characterized by a progressive decrease in hair density. AGA can occur in both men and women, but the incidence is higher in men. Male AGA primarily manifests as hair loss in the frontotemporal and vertex regions, presenting a typical "horseshoe" shape. Female AGA is characterized by thinning hair at the crown, without a receding hairline, and patients may also experience oily scalp, increased dandruff, and itching. Studies have shown that the incidence of AGA is positively correlated with age: the probability of developing the condition is 30% at age 30, 50% at age 50, and 80% at age 70. In recent years, due to the influence of environment, work stress, and unhealthy lifestyle and dietary habits, the onset of AGA is trending towards younger ages. While AGA is a chronic, progressive disease and not life-threatening, it is a disfiguring condition that severely impacts a patient's self-image, social interactions, and mental and emotional well-being.

[0003] There are currently many treatment options for AGA, including FDA-approved minoxidil and finasteride for treating both male and female pattern hair loss, but their mechanisms of action and routes of administration differ. Minoxidil is a calcium-based... 2+Calcium channel openers are effective in 80%-90% of hair loss patients, but adverse reactions can occur, such as dry and itchy skin, redness of the skin and hair, excessive facial hair in women, and contact dermatitis in rare cases. Finasteride, as a member of the azasteroid family, is used orally to treat male-onset AGA. Long-term use of finasteride can affect male sexual function, including decreased libido and erectile dysfunction. Local injection of platelet-rich plasma (PRP) improves microcirculation around hair follicles and promotes hair follicle regeneration. These growth factors may stimulate stem cells located in the dermal papilla ridges, promoting hair follicle growth; however, the use of PRP to treat AGA is still in its early stages. Androgen receptor inhibitors inhibit the transcription and translation of target genes by the complex formed by the binding of androgens and androgen receptors in dermal papilla cells, thereby inhibiting the formation of proteins with specific functions and affecting hair growth. Adverse reactions mainly include hyperkalemia, menstrual disorders, decreased libido, and breast tenderness. Hair transplantation, as a surgical method for treating AGA hair regrowth, involves surgically transplanting hair from one area of ​​the patient to another area of ​​the autologous or allogeneic body. However, the transplanted hair has specific requirements; hair loss typically occurs two months after surgery, and significant results are only visible after 6-9 months. The surgery requires highly skilled operators, is expensive, and has a low survival rate for transplanted hair follicles. Low-frequency energy laser therapy, as an adjunct treatment for AGA, uses low-frequency lasers to penetrate the epidermis, improve local blood circulation in the scalp, reduce hair follicle inflammation, and increase intracellular ATP for nutrition, but its mechanism of action remains unclear. In conclusion, finding a class of highly effective and low-toxicity drugs for treating AGA is essential. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the existing technology and avoid the side effects of existing commercially available drugs, the purpose of this invention is to provide the use of protein nano-selenium hydrogel in the preparation of drugs for the prevention and treatment of androgenetic alopecia. The use of protein nano-selenium hydrogel is safe and effective.

[0005] The objective of this invention is achieved through the following technical solution: The use of a protein-based selenium nanogel in the preparation of a drug for preventing and treating androgenetic alopecia, the preparation steps of which include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan, stirring the reaction at room temperature or 4°C for 3-12 h, removing unreacted substances by dialysis or centrifugation to obtain protein-based selenium nanoparticles; then, adding the protein-based selenium nanoparticles to the hydrogel matrix raw material, and preparing the protein-based selenium nanogel under thorough stirring and the action of a crosslinking agent or crosslinking inducer (such as triethanolamine to change pH or environmental changes).

[0006] The protein reducing agent is tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol, β-mercaptoethanol, cysteine, or glutathione.

[0007] The hydrogel matrix raw materials are carbomer gel, hyaluronic acid powder, chitosan, sodium alginate, or gelatin.

[0008] The albumin may be one or more of the following: human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin, donkey serum albumin, transferrin, or similar disulfide-rich proteins.

[0009] The protein nano-selenium hydrogel was prepared according to the following steps: (1) Dissolve albumin in a phosphate buffer solution of protein reducing agent with a pH of 5.0-9.0, with a final albumin concentration of 0.01-200 mg / mL and a final protein reducing agent concentration of 0.1-60 mM; react at 37℃ for 90-110 min, stirring to obtain a homogeneous protein solution with expanded spatial structure; then, add a sodium selenite solution or a sodium selenate solution containing chitosan to the homogeneous protein solution with expanded spatial structure, with a final chitosan concentration of 0.05-5 mg / mL and a final sodium selenite or sodium selenate concentration of 0.1-30 mM; stir and react at room temperature or 4℃ for 3-12 h to obtain a crude protein nano-selenium solution; place the crude protein nano-selenium solution in a dialysis bag and dialyze overnight with PBS solution at 0-20℃, or centrifuge the crude protein nano-selenium solution at 8000-30000 rpm for 10-60 minutes. The protein nanoparticles were obtained by repeatedly resuspending them in deionized water and centrifuging them several times. (2) Add carbomer gel to water and stir to make it fully swollen. Then add the protein nano-selenium particles and deionized water obtained in step (1). Add triethanolamine under full stirring to adjust the pH until it becomes a hydrogel, and obtain protein nano-selenium hydrogel. Alternatively, hyaluronic acid powder can be dissolved in water and stirred thoroughly to make it swell. Then, the protein nano-selenium particles obtained in step (1) and deionized water can be added. Finally, the cross-linking agents carbodiimide and N-hydroxysuccinimide can be added to cross-link hyaluronic acid and stirred thoroughly to make a hydrogel, thus obtaining a protein nano-selenium hydrogel. Alternatively, chitosan can be dissolved in acetic acid, and then the dissolved chitosan can be added to water. Next, the protein nano-selenium particles obtained in step (1) and deionized water can be added, and then the cross-linking agent glutaraldehyde can be added and stirred thoroughly to make it into a protein nano-selenium hydrogel.

[0010] Alternatively, sodium alginate solution can be added to crosslinking agent CaCl2 and stirred to prepare sodium alginate hydrogel, and then the protein nano-selenium particles obtained in step (1) and deionized water can be added to prepare protein nano-selenium hydrogel. Alternatively, gelatin can be ultrasonically dispersed in deionized water at 60°C, and the temperature induces cross-linking of gelatin molecules. Once the gelatin has completely formed a hydrogel, the protein nano-selenium particles obtained in step (1) and deionized water are added to prepare the protein nano-selenium hydrogel.

[0011] In step (1), the dialysis bag has a dialysis molecule rejection rate of no less than 1000.

[0012] The present invention has the following advantages and beneficial effects compared with the prior art: (1) The present invention uses protein nano-selenium hydrogel to prevent androgenetic alopecia, which has stronger antioxidant activity, higher safety and efficacy compared with existing inorganic nano-selenium hydrogel. Pharmacodynamic studies have shown that the protein nano-selenium hydrogel of the present invention has better efficacy in mouse models of androgenetic alopecia than inorganic nano-selenium hydrogel, with earlier melanin deposition and the ability to activate hair follicle cells to enter the hair growth phase earlier.

[0013] (2) The safe dosage range of selenium (Se) in the human body is very narrow, and it is easy to cause toxicity due to overdose. Although inorganic selenium nanoparticles improve the bioavailability and bioactivity of selenium and improve the safety of selenium supplementation, they still have problems such as easy oxidation and aggregation, which limit their widespread application. Human serum albumin (HSA) is a natural, non-toxic, non-immunogenic, biodegradable, and biocompatible drug carrier. It can be encapsulated in proteins through various drug binding sites in HSA molecules and transported to the treatment site, thereby improving the effective utilization of drugs. Therefore, this invention uses protein assembly technology to construct protein selenium nanoparticles (HSA-Se), and studies have found that it has better bioactivity and safety than inorganic selenium nanoparticles (Nano-Se).

[0014] (3) Previous research of this invention found that after preparing hydrogels from two types of nanoparticles, protein nano-selenium particles (HSA-Se) and inorganic nano-selenium particles (Nano-Se), the protein nano-selenium hydrogel (HSA-Se-Gel) has a significant promoting effect on AGA hair growth compared with the inorganic nano-selenium hydrogel (Nano-Se-Gel). The study found that the protein nano-selenium hydrogel (HSA-Se-Gel) has enzyme-mimicking activity, which can mimic the activity of CAT and SOD to clear ROS in the microenvironment around the hair follicle, inhibit the damage of excessive ROS to hair follicle cells, and promote hair regeneration. Attached Figure Description

[0015] Figure 1 These are morphological and characterization images of protein selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se), where A shows the macroscopic morphology of HSA-Se and Nano-Se particles; and B shows the transmission electron microscopy (TEM) images of HSA-Se and Nano-Se particles.

[0016] Figure 2 These are morphological and characterization images of protein-containing hydrogels (Gel), protein nano-selenium hydrogels (HSA-Se-Gel), inorganic nano-selenium hydrogels (Nano-Se-Gel), and blank carbomer gels (Matrix-Gel). A is the morphology image observed by the naked eye; B is the transmission electron microscope image; CJ is the rheological analysis of the gel, including amplitude scanning test, frequency scanning test, and linear viscoelastic region.

[0017] Figure 3 The in vitro biosafety of protein nano-selenium hydrogel (HSA-Se-Gel) and inorganic nano-selenium hydrogel (Nano-Se-Gel) is evaluated, where A is the biosafety evaluation of HSF cells and B is the biosafety evaluation of HaCaT cells.

[0018] Figure 4 This study evaluates the biocompatibility of mice after treatment with each gel group. AD represents the number of red blood cells (RBC), white blood cells (WBC), and platelets (PLT), and the total hemoglobin (HGB) level in mice treated with Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g), Nano-Se-Gel (Se 80 μg / g), HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), and Minoxidil on day 19. EH represents the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (SCr), and blood urea nitrogen (BUN) levels in mice on day 19 after treatment in each group. I represents the change in body weight of mice in each treatment group on day 19. J represents the H&E sections of major organs of mice in each treatment group on day 19.

[0019] Figure 5 This is a graph evaluating the antioxidant capacity of different forms of Se. A represents the DPPH scavenging rate of HSA-Se, Nano-Se, and VC (2.5 μg / ml); B represents the DPPH scavenging rate of different concentrations of HSA-Se, Nano-Se, and VC at 4 h; C represents the efficiency of HSA-Se, Nano-Se, and VC (20 μg / ml) in scavenging superoxide anions; D represents the concentration-dependent scavenging effect of HSA-Se on superoxide anions; E represents the scavenging effect of HSA-Se, Nano-Se, and VC (10 μg / ml) on hydroxyl radicals; and F represents the concentration-dependent scavenging effect of HSA-Se on hydroxyl radicals.

[0020] Figure 6The figures show the effects of HSA-Se and Nano-Se on reducing H2O2-induced oxidative stress in HSF cells. In the figure, A represents cell viability after 2 h of treatment with different concentrations of H2O2; BC represents the protective effects of different concentrations of HSA-Se and Nano-Se against H2O2-induced oxidative stress damage; D represents the expression of ROS in HSF cells after treatment with different concentrations of HSA-Se and Nano-Se; and E represents the quantification of ROS fluorescence intensity in HSF cells.

[0021] Figure 7 The results show the evaluation of hair regeneration in AGA mice using HSA-Se and Nano-Se, where A represents the overall hair regeneration status of AGA mice; B represents the length of newly grown hair; C represents the diameter of newly grown hair; D represents the weight of newly grown hair / area of ​​the bald area; E represents the skin color score of AGA mice; F represents the H&E section of AGA mouse skin; and G represents the MDA content in the skin of AGA mice. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0023] Example 1: Preparation of protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) and their hydrogels (HSA-Se-Gel and Nano-Se-Gel). (1) Preparation of protein nano-selenium (HSA-Se): Human serum albumin was dissolved in phosphate-buffered saline (PBS) containing tris(2-carboxyethyl)phosphine (TCEP) at pH 5.0, with a final albumin concentration of 10 mg / mL and a final TCEP concentration of 20 mM. The mixture was reacted at 37 °C for 100 min with stirring to obtain a homogeneous protein solution with expanded spatial structure. Then, a sodium selenite solution containing chitosan was added to the homogeneous protein solution with expanded spatial structure, with a final chitosan concentration of 0.05 mg / mL and a final sodium selenite concentration of 5 mM. The mixture was stirred at 4 °C for 12 h to obtain a crude protein nano-selenium solution. The crude protein nano-selenium solution was placed in a dialysis bag and dialyzed overnight in PBS solution at a low temperature of 0–20 °C to finally obtain protein nano-selenium particles (HSA-Se).

[0024] (2) Preparation of inorganic nano-selenium (Nano-Se): Weigh 46.05 mg of glutathione (GSH) and 30 mg of human serum albumin (BSA) and pour them into 7.0575 ml of water. After stirring evenly, add 0.3225 mL of Na2SeO3 solution with a concentration of 20 mg / mL, and finally add 120 μL of NaOH solution with a concentration of 1 mol / L. Stir evenly to prepare an inorganic nano-selenium particle (Nano-Se) solution.

[0025] (3) Preparation of HSA-Se and Nano-Se carbomer hydrogels: First, 3 g of carbomer 934 was added to 100 mL of water and stirred thoroughly to swell, thus preparing a 3% carbomer hydrogel. Then, 13 mL of 3% carbomer 934 was added to 25.675 mL of deionized water and stirred thoroughly to swell. 1.33 g of HSA-Se obtained in step (1) and Nano-Se obtained in step (2) were added. At the same time, no substance was added as a blank group. Under thorough stirring, a cross-linking inducer (triethanolamine) was added to prepare a protein-only gel (Gel), a 60 μg / g protein nano-selenium carbomer hydrogel (HSA-Se-Gel), an inorganic nano-selenium carbomer hydrogel (Nano-Se-Gel), and a blank carbomer gel (Matrix-Gel).

[0026] (4) Preparation of HSA-Se and Nano-Se chitosan hydrogels: First, weigh 1.42 g of chitosan, add 2% acetic acid solution and stir thoroughly to dissolve, allowing the chitosan to swell completely; then add 1.33 g of HSA-Se obtained in step (1) and Nano-Se obtained in step (2). Stir thoroughly until homogeneous; finally, add 2% glutaraldehyde and continue stirring to obtain 60 μg / g protein nano-selenium chitosan hydrogel (HSA-Se-Gel) and inorganic nano-selenium chitosan hydrogel (Nano-Se-Gel).

[0027] (5) Preparation of HSA-Se and Nano-Se hyaluronic acid hydrogels: First, sodium hyaluronate was dissolved thoroughly in 0.01 M HCl. 100 mg of acidic HA was activated in 50 mL DMSO for 24 h with 251 mg carbodiimide (EDC) and 151 mg N-hydroxysuccinimide (NHS). Then, 1.33 g of HSA-Se obtained in (1) and Nano-Se obtained in (2) were added and stirred thoroughly. 60 μg / g protein nano-selenium hyaluronic acid hydrogel (HSA-Se-Gel) and inorganic nano-selenium hyaluronic acid hydrogel (Nano-Se-Gel) were obtained.

[0028] Example 2: Characterization of protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) The macroscopic morphology of the protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) obtained in Example 1 is as follows: Figure 1 As shown in Figure A; protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) were observed using transmission electron microscopy. Figure 1 As shown in B.

[0029] Example 3: Characterization of protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) hydrogels The morphologies of the protein-containing gel (Gel), protein nano-selenium hydrogels (HSA-Se-Gel, protein nano-selenium carbomer hydrogel), inorganic nano-selenium hydrogels (Nano-Se-Gel, inorganic nano-selenium carbomer hydrogel), and blank carbomer gel (Matrix-Gel) obtained in step (3) of Example 1 were observed, such as Figure 2 As shown in Figure A, HSA-Se-Gel and Nano-Se-Gel appeared orange-red, with the color deepening as the concentration increased, while Matrix-Gel appeared transparent white. Transmission electron microscopy observation of the morphology of protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) hydrogels revealed a loose and porous structure, as shown in Figure A. Figure 2 As shown in B. Amplitude scanning, frequency scanning, and linear viscoelastic region measurements using a rheometer indicated that the gels were more elastic than viscous, easily spread and retained on the skin, making them suitable for topical application, such as... Figure 2 As shown in CJ.

[0030] Example 4: In vitro biosafety evaluation of protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) obtained in Example 1. Methods: HSF cells (5×10⁻⁶) were used to prepare the HSF cells. 3Inoculate each well with HSA-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 μM) and Nano-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 μM), with 5 replicates per group. After 24 h, discard the culture medium and add 100 μL of serum-free medium containing 20 μL MTT to each well. After 4 h, add 100 μL of SDS to each well. After the blue-purple crystalline formazan dissolves for 24 h, measure the absorbance at 570 nm. Calculate cell viability: Cell viability % = (OD value of drug-treated group - OD value of blank group) / OD value of blank group × 100%.

[0031] Conclusion: Treatment of HSF and HaCaT cells with different concentrations of HSA-Se and Nano-Se for 24 h, and cell viability as determined by MTT assay, revealed that neither HSA-Se nor Nano-Se exhibited significant cytotoxicity below 10 μM (e.g., Figure 3 (A and B).

[0032] Example 5: Effects and in vivo safety of the protein nano-selenium hydrogel (HSA-Se-Gel, protein nano-selenium carbomer hydrogel) and inorganic nano-selenium hydrogel (Nano-Se-Gel, inorganic nano-selenium carbomer hydrogel) obtained in step (3) of Example 1 on AGA mouse model. 1. Establish the model: Seven-week-old male C57BL / 6 mice, weighing approximately 20 g, were used as experimental subjects. Mice were randomly divided into groups, and hair was removed from a 2×3 cm area of ​​the mouse's back using a razor and depilatory cream. All mice were artificially induced to enter the telogen phase, resulting in pink skin. The mice were then treated daily with 1 mL of a 0.5% (w / v) testosterone ethanol solution (in which testosterone was dissolved in 50% ethanol).

[0033] 2. Grouping and administration: Mice were randomly assigned to groups and treated with the following drugs: Normal, blank carbomer gel, Model, Minoixdil (5%, 50 mg / g), HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g), and Nano-Se-Gel (Se 80 μg / g). Testosterone was applied to each group every morning, and 0.2 mL of each drug was applied 4 hours later.

[0034] 3. Observation: (1) Hair growth: Photos were taken daily to record the time it took for the skin of AGA mice to darken and the hair growth. On the 19th day of the experiment, the newly grown hair of AGA mice was taken with a razor, and the diameter, length and weight of the newly grown hair of each group of AGA mice were recorded.

[0035] (2) Oxidative markers in mouse skin: MDA: On day 19 after hair removal, mice were sacrificed, and skin from the hair-removed area on the back of the mice was taken, weighed, and recorded. The skin was stored at -80 ℃. The grinding adapter and steel balls were pre-frozen overnight at -20 ℃. The skin from the -80 ℃ freezer was taken, minced, and placed in a 2 mL special centrifuge tube. Two No. 5 steel balls were added, along with 500 μL of PBS. Note: The weight ratio of skin tissue to PBS was 1:9. The tissue was ground at 65 Hz for 1 min in a tissue homogenizer, and the steel balls were carefully removed. The tissue was then centrifuged for 30 min (4 ℃, 15000 rpm) in a refrigerated centrifuge, and the supernatant was collected. The MDA content was determined according to the MDA kit instructions.

[0036] (3) Biosafety: On day 19, the heart, liver, spleen, lung and kidney of mice were taken, fixed with 4% paraformaldehyde and stained with H&E.

[0037] 4. Conclusion: AGA mice were percutaneously treated with HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g), and Nano-Se-Gel (Se 80 μg / g) for 19 days. Mice were then sacrificed for complete blood count, liver and kidney function tests, and weight changes. Pathological sections of the heart, liver, spleen, lungs, and kidneys of each group of mice were also performed. The results showed that HSA-Se-Gel had good biocompatibility. Figure 4 (AJ).

[0038] Example 6: In vitro antioxidant evaluation of protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) obtained in Example 1. 1. Experimental Methods DPPH method for determining free radicals: (1) Prepare a 0.1 mM DPPH ethanol solution: Dissolve 0.002 g DPPH in 50 mL of anhydrous ethanol and store in the dark.

[0039] (2) Prepare a 0.5 mg / mL VC solution.

[0040] (3) Prepare sample solutions: inorganic nano selenium (0.625, 1.25, 2.5, 5 μg / mL), protein nano selenium (0.625, 1.25, 2.5, 5 μg / mL).

[0041] (4) Mix the sample solution and VC solution with DPPH ethanol solution respectively (in the dark), place at room temperature in the dark, and measure the UV absorbance at 517 nm at 0.5 h, 1 h, 2 h and 4 h.

[0042] (5) Group setting: Sample group (A) i ): Sample solution (100 μL) + DPPH ethanol solution (100 μL); Blank group (A) j ): Sample solution (100 μL) + anhydrous ethanol (100 μL); Control group (A0): deionized water (100 μL) + DPPH ethanol solution (100 μL); 3 parallel samples per group.

[0043] (6) Calculation of results: DPPH clearance rate % = [1 - (A i -A j ) / A0]×100%; DPPH residual rate%=(A i -A j ) / A0×100%.

[0044] Saffron Red T Determination of Hydroxyl Radicals: (1) Prepare sample solutions HSA-Se and Nano-Se (2.5, 5, 10 μg / mL), with 3 parallel samples in each group.

[0045] (2) Prepare a 25% ethanol solution as a control.

[0046] (3) Prepare 0.30% H2O2 solution; 0.40 mmol / L MnSO4 solution; 0.20 mol / L H2SO4; 0.030% saffron T solution.

[0047] (4) Add 0.16 mL, 0.07 mL, 0.08 mL, and 0.12 mL of MnSO4 solution, H2SO4 solution, saffron red T solution, and H2O2 solution to a 1.5 mL centrifuge tube, respectively. Dilute with distilled water to the 1 mL mark, shake well, and let stand in the dark for 10 min. Using distilled water as a reference, measure the absorbance at a wavelength of 518 nm and calculate the difference ΔA between the two absorbance values.

[0048] (5) Add different concentrations of HSA-Se and Nano-Se to the above system, and perform the same operation as above to measure the absorbance A of the added antioxidant solution. s Saffron red T and sulfuric acid were used as blank systems, with an absorbance of A0; the absorbance of the hydroxyl radical reaction system without antioxidants was A; each group had 3 parallel samples.

[0049] (6) The hydroxyl radical scavenging rate can be expressed as: R = (A s -A ) / (A0-A )×100%.

[0050] Determination of pyrogallol by superoxide anion: (1) Prepare sample solutions HSA-Se and Nano-Se (5, 10, 20 μg / mL), with 3 parallel samples in each group.

[0051] (2) Prepare 7.567 mg / mL pyrogallol (dissolved in 1 mM HCl), Tris-HCl, 1 mM HCl, and 0.1 M HCl.

[0052] (3) Add the sample, Tris-HCl, and pyrogallol in sequence, and measure the absorbance at a wavelength of 319 nm. Measure once every 30 s for 240 s. Use Tris-HCl and 1 mM HCl as blank systems.

[0053] The total antioxidant capacity of HSA-Se and Nano-Se was evaluated using the DPPH method. The results showed that HSA-Se had a stronger total antioxidant capacity, which was concentration-dependent and significantly better than Nano-Se. Figure 5 The pyrogallol method was used to evaluate the scavenging ability of different selenium forms and vitamin C for superoxide anions. The study found that HSA-Se and the positive control vitamin C significantly reduced the generation of superoxide anions, while having almost no effect on Nano-Se, suggesting that HSA-Se is significantly more effective than Nano-Se in scavenging superoxide anions. Figure 5 The scavenging rate of HSA-Se and Nano-Se on hydroxyl radicals showed a concentration-dependent effect. Furthermore, the scavenging rate results of HSA-Se and Nano-Se on hydroxyl radicals indicated that HSA-Se had a significantly higher scavenging rate than Nano-Se. Figure 5The EF), and its clearance rate is positively correlated with its concentration.

[0054] Example 7: In vitro cell experiments on protein-based selenium nanoparticles (HSA-Se) and inorganic selenium nanoparticles (Nano-Se) obtained in Example 1 1. H2O2-induced oxidative stress (1) HSF cells (5×10 3 Cells were seeded in 96-well plates at concentrations of 200, 400, and 800 μM for 24 h. Different concentrations of H2O2 (200, 400, and 800 μM) were added in five replicates per group. After 2 h of treatment, the culture medium was discarded, and 100 μL of serum-free medium containing 20 μL of MTT was added to each well. After 4 h, 100 μL of SDS was added to each well. The absorbance was measured at 570 nm after the blue-purple formazan crystals dissolved for 24 h.

[0055] (2) HSF cells (5×10 3 (Number of cells / well) were seeded into 96-well plates. After 24 h of adhesion, different concentrations of HSA-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 μM) and Nano-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 μM) were added, with 5 replicates per group. After 24 h of treatment, 800 μM H2O2 was added. After 2 h of treatment, the culture medium was discarded, and 20 μL of MTT was added to each well. After 4 h, 100 μL of SDS was added to each well. After 24 h, the absorbance was measured at 570 nm.

[0056] 2. Detection of ROS in HSF cells – DCFH-DA fluorescent probe method (1) HSF cells (1×10 5 (Numbers / well) were seeded into 12-well plates. After 24 h of adhesion, 100 μM H2O2 was added for 1 h of treatment, followed by HSA-Se (1.25 μM, 0.3125 μM) and Nano-Se (1.25 μM, 0.3125 μM) treatment for 24 h.

[0057] (2) Remove the cells, wash them once with PBS, add 10 μM of DCFH-DA fluorescent probe, incubate at 37℃ for 30 min, and photograph the fluorescence under an inverted fluorescence microscope.

[0058] 3. Conclusion: HSF cells were treated with 100, 200, 400, 600, and 800 μM H2O2 for 2 h. Cell viability was assessed using the MTT assay, revealing that the cell death rate was approximately 50% at 800 μM. Subsequent experiments used 800 μM H2O2 as the concentration to induce oxidative stress damage in HSF cells. Figure 6 (A). Cells were first damaged with 800 μM H2O2 for 2 h, and then treated with different concentrations of HSA-Se. The results showed that HSA-Se had a protective effect on cells starting at 1.25 μM, and HSA-Se had a protective effect against H2O2-induced oxidative stress damage to cells. Figure 6 (B). Furthermore, after pretreatment with different concentrations of HSA-Se and Nano-Se for 24 h, followed by H2O2 injury to cells for 2 h, the results showed that HSA-Se had a stronger protective effect against oxidative stress damage compared to Nano-Se. Figure 6 (C). We also detected intracellular ROS levels using the DCFH-DA fluorescent probe. After treatment with 100 μM H2O2, cells were given HSA-Se (0.3125, 1.5 μM) and Nano-Se (0.3125, 1.5 μM). Incubation with the DCFH-DA fluorescent probe revealed that HSA-Se significantly reduced intracellular ROS levels. Although Nano-Se had lower fluorescence intensity than the model group, HSA-Se showed a more significant effect in reducing intracellular ROS. Figure 6 (DE).

[0059] Example 8: Effects of the protein nano-selenium hydrogel (HSA-Se-Gel, protein nano-selenium carbomer hydrogel) and inorganic nano-selenium hydrogel (Nano-Se-Gel, inorganic nano-selenium carbomer hydrogel) obtained in step (3) of Example 1 on the AGA mouse model. 1. Establish the model: Seven-week-old male C57BL / 6 mice, weighing approximately 20 g, were used as experimental subjects. Mice were randomly divided into groups, and hair was removed from a 2×3 cm area of ​​the mouse's back using a razor and depilatory cream. All mice were artificially induced to enter the telogen phase, resulting in pink skin. The mice were then treated daily with 1 mL of a 0.5% (w / v) testosterone ethanol solution (in which testosterone was dissolved in 50% ethanol).

[0060] 2. Grouping and administration: Mice were randomly assigned to groups and treated with the following drugs: Normal, blank carbomer gel, Model, Minoixdil (5%, 50 mg / g), HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g), and Nano-Se-Gel (Se 80 μg / g). Testosterone was applied to each group every morning, and 0.2 mL of each drug was applied 4 hours later.

[0061] 3. Observation: (1) Hair growth: Photos were taken daily to record the time it took for the skin of AGA mice to darken and the hair growth. On the 19th day of the experiment, the newly grown hair of AGA mice was taken with a razor, and the diameter, length and weight of the newly grown hair of each group of AGA mice were recorded.

[0062] (2) Oxidative markers in mouse skin: MDA: On day 19 after hair removal, mice were sacrificed, and skin from the hair-removed area on the back of the mice was taken, weighed, and recorded. The skin was stored at -80 ℃. The grinding adapter and steel balls were pre-frozen overnight at -20 ℃. The skin from the -80 ℃ freezer was taken, minced, and placed in a 2 mL special centrifuge tube. Two No. 5 steel balls were added, along with 500 μL of PBS. Note: The weight ratio of skin tissue to PBS was 1:9. The tissue was ground at 65 Hz for 1 min in a tissue homogenizer, and the steel balls were carefully removed. The tissue was then centrifuged for 30 min (4 ℃, 15000 rpm) in a refrigerated centrifuge, and the supernatant was collected. The MDA content was determined according to the MDA kit instructions.

[0063] 4. Conclusion: An AGA model was established using C57BL / 6 mice. The mice were divided into groups and treated with the following medications: carbomer gel (containing only protein), Model, Minoixdil (5%, 50 mg / g), HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g), and Nano-Se-Gel (Se 80 μg / g). The Normal group received no treatment. On the first day of hair removal, all mice had pink skin with no obvious vellus hair growth. On day 9, melanin deposition began to appear in the skin of the control group, while no melanin deposition was observed in the other groups. By day 11, noticeable hair growth had appeared in the control group. Furthermore, the HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), and HSA-Se-Gel (Se 80 μg / g) groups all showed signs of hair growth on day 11. The skin darkening of the mice in these groups began earlier than in the Nano-Se-Gel group, where melanin deposition appeared earlier. In contrast, the positive control drug minoxidil only showed melanin deposition after 13 days. At day 19, the regenerated hair coverage rate in the HSA-Se-Gel group reached over 90%, but the Nano-Se-Gel (Se 20 μg / g) and Nano-Se-Gel (Se 40 μg / g) groups did not reach over 90% hair coverage. Figure 7 (A). Newly grown hair was removed with a razor, and the hair length, diameter, and weight were measured for each group of mice. The results showed that the hair length, diameter, and weight of the HSA-Se-Gel group were all superior to those of the Nano-Se-Gel group, and the HSA-Se-Gel (Se 40 μg / g) group had better hair regeneration effect than the HSA-Se-Gel (Se 20 μg / g) group and the HSA-Se-Gel (Se 80 μg / g) group. Figure 7 (BD). Skin scores in mice also showed that melanin deposition occurred earlier in the HSA-Se-Gel group than in the Nano-Se-Gel group, indicating that the HSA-Se-Gel group entered the hair growth phase earlier. Figure 7 E). Furthermore, H&E sections of skin from AGA mice ( Figure 7As can be seen from F), compared to the model group, the number of hair follicles in the HSA-Se-Gel group was significantly increased, and complete hair follicle structures were visible, with most follicles in the anagen or early catagen phase. The miniaturization and atrophy of hair follicles observed in the model group were significantly improved after HSA-Se-Gel administration. Furthermore, in the HSA-Se-Gel group, most hair bulbs were located in the subcutaneous fat layer, and the skin thickness also increased after HSA-Se-Gel administration. Although the Nano-Se-Gel group showed improvement compared to the model group, its effect was far less than that of the HSA-Se-Gel group in treating AGA. After administration, the MDA content in the mouse skin was lower than that in the model group (…). Figure 7 (G).

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a protein nano-selenium hydrogel in the preparation of drugs for preventing and treating androgenetic alopecia, characterized in that: The preparation steps of the protein nano-selenium hydrogel include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan, stirring at room temperature or 4°C for 3-12 h, removing unreacted substances by dialysis or centrifugation to obtain protein nano-selenium particles; then adding the protein nano-selenium particles to the hydrogel matrix raw material, and preparing the protein nano-selenium hydrogel under thorough stirring and the action of a crosslinking agent or crosslinking inducer; the protein reducing agent is tris(2-carboxyethyl)phosphine; the hydrogel matrix raw material is carbomer gel.

2. The use of the protein nano-selenium hydrogel according to claim 1 in the preparation of drugs for preventing and treating androgenetic alopecia, characterized in that: The protein nano-selenium hydrogel was prepared according to the following steps: (1) Dissolve albumin in a phosphate buffer solution of protein reducing agent with a pH of 5.0-9.

0. The final concentration of albumin is 0.01-200 mg / mL and the final concentration of protein reducing agent is 0.1-60 mM. React at 37℃ for 90-110 min, stir, and obtain a homogeneous protein solution with expanded spatial structure. Then, add sodium selenite solution containing chitosan to the homogeneous protein solution with expanded spatial structure. The final concentration of chitosan is 0.05-5 mg / mL and the final concentration of sodium selenite is 0.1-30 mM. Stir and react at room temperature or 4℃ for 3-12 h to obtain a crude protein nano-selenium solution. Place the crude protein nano-selenium solution in a dialysis bag and dialyze overnight with PBS solution at 0-20℃, or centrifuge the crude protein nano-selenium solution at 8000-30000 rpm for 10-60 min and repeatedly resuspend in deionized water and centrifuge several times to finally obtain protein nano-selenium particles. (2) Add carbomer gel to water and stir to make it fully swell. Then add the protein nano-selenium particles obtained in step (1) and deionized water. Add triethanolamine under full stirring to induce carbomer crosslinking into hydrogel to obtain protein nano-selenium hydrogel.

3. The use according to claim 2, characterized in that: In step (1), the dialysis bag has a dialysis molecule retention of no less than 1000.

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