Preparation method of hydrogen-producing liposome, hydrogen-producing liposome, and applications thereof in hydrogen-producing, anti-inflammatory, and anti-hair loss products

Abstract

The present invention discloses a preparation method of hydrogen-producing liposomes, hydrogen-producing liposomes, and their applications in hydrogen-producing, anti-inflammatory, and anti-hair loss products. The method of the present invention prepares stable and dense liposomes through a simple hydration method, with a reaction time of only 15 minutes and a required temperature of only 45-50 °C. The synthesized liposomes have good dispersibility, strong stability, and good encapsulation. The preparation method has a simple process, mild reaction conditions, short reaction time, low energy consumption, and can achieve large-scale production. In the subsequent reaction of encapsulating antioxidants and Au / Pt / Ag NPs, the synthesized liposomes are mixed with the encapsulant and sonicated in an ice bath to obtain a hydrogen-producing system with completed assembly. The reaction conditions are simple, fast, and have a high success rate, thus opening up new opportunities for hydrogen-producing systems based on Au, Pt, and Ag nanoparticles in the fields of biomedical and cosmetic anti-inflammatory and anti-aging.

Description

Technical Field

[0001] The present invention belongs to the technical field of liposomes, and specifically relates to a preparation method of hydrogen-producing liposomes, hydrogen-producing liposomes, and their applications in hydrogen-producing, anti-inflammatory, and anti-hair loss products. Background Art

[0002] As a potential antioxidant, hydrogen (H2) can selectively reduce highly cytotoxic ROS in diseased cells. However, due to the low solubility of H2 in body fluids, the efficiency of delivering H2 by traditional administration routes is extremely low. The therapeutic effect of hydrogen therapy mainly depends on the concentration of hydrogen in the diseased tissue. Hydrogen itself has low solubility and high volatility, making it difficult to maintain a high concentration at the site to be treated for a long time.

[0003] The structure of liposomes is surrounded by hydrophilic heads on the outside and connected in the form of a phospholipid bilayer sphere with hydrophobic tails on the inside. This spherical shape has the advantage of capturing all bipolar materials, such as hydrophilic and hydrophobic materials, which provides flexibility for encapsulating hydrophilic and hydrophobic drugs. Therefore, using lipids as composite materials makes liposomes biocompatible and can be used in various applications such as cosmetics, medicine, and drug delivery.

[0004] Due to their excellent physical and chemical properties, metal nanoparticles have been widely used in various fields. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a preparation method of hydrogen-producing liposomes, which includes,

[0007] Preparing a nanoparticle solution: adding a gold salt, a platinum salt, or a silver salt to a solvent, adding a stabilizer and a reducing agent for reaction, and obtaining a nanoparticle solution after dialysis;

[0008] Preparing liposomes containing a bioactive compound; the bioactive compound includes one or more of lutein, vitamin E, coumarin, carotenoid, resveratrol, lycopene, riboflavin, curcumin, emodin;

[0009] Preparing hydrogen-producing liposomes: mixing the liposomes containing the bioactive compound, an antioxidant, and the nanoparticle solution and then performing ultrasonic treatment to obtain hydrogen-producing liposomes; the antioxidant includes one or more of ascorbic acid, quercetin, anthocyanin, sodium isoascorbate, baicalein, catechin, vitamin C phosphate, ascorbyl palmitate, ascorbyl glucoside, 2-o-ethyl ascorbic acid, 3-O-ethyl ascorbic acid ether, sodium sulfite.

[0010] As a preferred embodiment of the preparation method of the hydrogen-producing liposomes of the present invention: the gold salt includes chloroauric acid, and the platinum salt includes chloroplatinate; the concentration of the gold salt, platinum salt or silver salt is 0.05-5 mM.

[0011] As a preferred embodiment of the preparation method of the hydrogen-producing liposomes of the present invention: the solvent includes water, the temperature for the reaction is 0-8 °C, and the time is 0.5-2 h.

[0012] As a preferred embodiment of the preparation method of the hydrogen-producing liposomes of the present invention: the stabilizer includes Tween 80, and the concentration of Tween 80 is 0.01-1 wt%; the reducing agent includes sodium borohydride, and 20% of 1-10 mg / mL of sodium borohydride is added; the dialysis time is 6-24 h.

[0013] As a preferred embodiment of the preparation method of the hydrogen-producing liposomes of the present invention: the liposomes include several of dipalmitoyl phosphatidylcholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, Tween, sodium deoxycholate, polyethylene glycol 2000, octadecylamine, sodium taurocholate, polyoxyethylene polymer, dioleoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine polyethylene glycol 2000.

[0014] As a preferred embodiment of the preparation method of the hydrogen-producing liposomes of the present invention: dipalmitoyl phosphatidylcholine, cholesterol, Tween, polyoxyethylene polymer and bioactive compound are mixed and dissolved in a solvent, the solvent is removed, and the product is dissolved in deionized water to obtain liposomes with a concentration of 0.02-2 wt%; wherein the concentration of the bioactive compound is 0.0001-0.005%; wherein, the mass ratio of dipalmitoyl phosphatidylcholine, cholesterol, Tween, and polyoxyethylene polymer is 15-25:4-5:0.3-5:3-8.

[0015] As a preferred embodiment of the preparation method of the hydrogen-producing liposomes of the present invention: the removal of the solvent includes rotary evaporation to remove the solvent, and the temperature is 45 °C.

[0016] As a preferred embodiment of the preparation method of the hydrogen-producing liposomes of the present invention: in step 3, the mass ratio of the liposomes containing the bioactive compound to the nanoparticle solution is 1:0.5-2; the concentration of the antioxidant is 0.5-20 wt%.

[0017] Advantages of the present invention: The present invention constructs a hydrogen production system in liposomes, providing unprecedented insights for the preparation of a stable and efficient hydrogen production system. Through liposome encapsulation technology, specific oxidants and gold nanoparticles are encapsulated into liposomes embedded with bioactive compounds to construct a closely fitting hydrogen production nanoreactor, which can effectively reduce the ultraviolet absorption peak of methylene blue at 664 nm under natural white light irradiation, creating a new type of hydrogen production system.

[0018] The method described in the present invention prepares stable and dense liposomes through a simple hydration method. The reaction time is only 15 minutes, and the required temperature is only 45 - 50 °C. The synthesized liposomes have good dispersibility, strong stability, and good encapsulation. The preparation method has a simple process, mild reaction conditions, short reaction time, low energy consumption, and can be mass-produced. In the subsequent reaction of encapsulating antioxidants and Au / Pt / Ag NPs, the synthesized liposomes are mixed with the encapsulant and ultrasonically treated in an ice bath to obtain the assembled hydrogen production system. The reaction conditions are simple, fast, and have a high success rate, thus opening up new opportunities for hydrogen production systems based on Au, Pt, and Ag nanoparticles in the fields of biomedicine and anti-inflammatory and anti-aging of cosmetics. Brief Description of the Drawings

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Among them:

[0020] Figure 1 It is a transmission electron microscope image of the particle size distribution and a particle size analysis chart in the examples of the present invention.

[0021] Figure 2 It is an image of the synthesized liposomes observed under a microscope in the examples of the present invention.

[0022] Figure 3 It is the ultraviolet spectrum of the methylene blue MB solution before and after adding HPS under white light irradiation.

[0023] Figure 4 It is the ultraviolet spectrum of pure methylene blue MB under white light irradiation (upper figure) and the ultraviolet spectrum of the methylene blue MB solution within 10 minutes without light irradiation after adding HPS (lower figure).

[0024] Figure 5 It is the ultraviolet absorption spectrum of the MB solution with and without light irradiation after adding liposomes encapsulating AA, Au NPs, liposomes, and HPS (upper figure) and the ultraviolet absorption spectrum of the MB solution with and without light irradiation after adding liposomes without photosensitizer encapsulating AA and Au NPs, lutein liposomes, lutein liposomes encapsulating Au NPs, and lutein liposomes encapsulating AA and HPS (lower figure).

[0025] Figure 6is the survival rate of RAW264.7 cells after adding HPS at various concentrations.

[0026] Figure 7 are the microscope pictures of Raw264.7 cells stained with MB under different conditions: (a) untreated; (b) added with HPS; (c) irradiated with light after adding HPS.

[0027] Figure 8 are the fluorescence microscope pictures of Raw264.7 cells stained with DCFH-DA under different conditions.

[0028] Figure 9 DCF intensity of ROS produced by LPS-induced RAW264.7 cells after different treatments.

[0029] Figure 10 (left figure) is the IL-6 level of LPS-induced RAW264.7 cells after different treatments; (right figure) is the TNF-α level of LPS-induced RAW264.7 cells after different treatments.

[0030] Figure 11 is the cell proliferation of human dermal papilla cells after adding HPS at various concentrations with light irradiation.

[0031] Figure 12 are the UV pictures of the hydrogen-producing liposome containing riboflavin before and after white light irradiation after adding MB solution.

[0032] Figure 13 are the UV pictures of the hydrogen-producing liposome containing resveratrol before and after white light irradiation after adding MB solution.

[0033] Figure 14 are the UV pictures of the hydrogen-producing liposome containing sodium ascorbate before and after white light irradiation after adding MB solution.

[0034] Figure 15 are the UV pictures of the hydrogen-producing liposome containing vitamin C ethyl ether before and after white light irradiation after adding MB solution.

[0035] Figure 16 is the cell proliferation of human dermal papilla cells after adding HPS at various concentrations without light irradiation.

[0036] Figure 17 are the optical microscope pictures of human dermal papilla cells stained with β-galactosidase.

[0037] Figure 18 are the fluorescence microscope pictures of human dermal papilla cells stained with DCFH-DA before and after light irradiation (left figure), and the DCF intensity of ROS produced by H2O2-induced human dermal papilla cells (right figure).

[0038] Figure 19IL-1α levels (left panel) and IL-6 levels (right panel) in human dermal papilla cells induced by dihydrotestosterone before and after light exposure. Detailed implementation manners

[0039] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following specific embodiments are used to describe the detailed implementation manners of the present invention in detail.

[0040] Example 1:

[0041] Step 1, Preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath. Deionized water was added to the flask and stirred rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. 0.5 mL of the aqueous chloroauric acid solution was taken, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and the mixture was stirred for 20 minutes. Then, 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL was gradually added dropwise. After 1 h, the liquid was placed in a 14 kDa MW dialysis bag and dialyzed with deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, which was stored at 4 °C in a refrigerator.

[0042] Step 2, Preparation of lutein-containing liposomes: 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween 80, 18 mg of polyethylene oxide polymer (average molecular weight 2000, CAS No.: 25322-68-3), and 2 mg of lutein were mixed and dissolved in 60 mL of ethanol, and the mixture was placed in a rotary evaporator. At 45 °C, the organic solvent was removed, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0043] Step 3, Preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of the liposomes obtained in Step 2, 1.2 g / L of L-ascorbic acid, and 10 mL of the aqueous solution of Au nanoparticles obtained in Step 1 were ultrasonically hydrated in an ice bath for 30 min to obtain hydrogen-producing liposomes encapsulating Au nanoparticles (hereinafter referred to as HPS).

[0044] Figure 1 Particle size analysis of AuNPs was performed using TEM transmission electron microscopy. The Au nanoparticle suspension was deposited on a 400-mesh carbon film copper grid to take TEM images. Approximately 50 individual particles were calculated from the taken TEM images, and the average diameter (Dc) of each AuNPs sample was calculated. The results showed that the synthesized Au NPs had a particle size mostly in the range of 2-3 nm and were uniformly and densely dispersed.

[0045] Figure 2The hydrogen-producing liposomes (hereinafter referred to as HPS) encapsulated with Au nanoparticles were observed using a microscope. The liquid to be observed was smeared on a glass slide and placed under a hot stage polarizing microscope. HPS was observed at 10*50 magnification and images were taken. The results showed that HPS was evenly dispersed under the microscope, with dense vesicles and relatively uniform sizes.

[0046] Figure 3 The ultraviolet absorption analysis of methylene blue (MB) solution before and after adding HPS under white light irradiation was carried out using an ultraviolet spectrophotometer. 18.6 mg of methylene blue powder was weighed and dissolved in 50 mL of deionized water to prepare a test solution. The test was carried out using an ultraviolet spectrophotometer. 2 mL of deionized water and 1 mL of HPS solution were added to the blank cell, and 1.5 mL of deionized water, MB solution and 1 mL of HPS solution were added to the sample cell. The ultraviolet absorption peaks of the sample in the range of 500 - 800 nm were detected at an interval of 1 nm. The sample cell was tested once when it was just placed, and then tested again after irradiating the sample cell and the blank control cell with white light for 2 min simultaneously. The results showed that for the hydrogen-producing liposomes (HPS) synthesized in step 3 of the example, after adding methylene blue (MB) solution (HPS group), the absorption of MB at 664 nm did not decrease significantly. However, after white light irradiation (HPS+L group), the absorption decreased sharply, indicating that after light irradiation, HPS successfully produced hydrogen and reduced MB in the solution. Figure 3 Among them, the HPS group was the light absorption of HPS and methylene blue MB without light irradiation at 664 nm, the MB group was the light absorption of methylene blue at 664 nm, and the HPS+L group was the light absorption of HPS and methylene blue MB with light irradiation at 664 nm.

[0047] Figure 4 The ultraviolet absorption analysis of pure methylene blue MB under white light irradiation (upper figure) and methylene blue MB solution added with HPS without light irradiation (lower figure) was carried out within 10 min using an ultraviolet spectrophotometer.

[0048] 18.6 mg of methylene blue powder was weighed and dissolved in 50 mL of deionized water to prepare a test solution. The test was carried out using an ultraviolet spectrophotometer. Deionized water was added to the blank cell and MB solution was added to the sample cell. The ultraviolet absorption peaks of the sample in the range of 500 - 800 nm were detected at an interval of 1 nm. The sample cell was tested once when it was just placed, and then tested respectively after different times of white light irradiation of the sample cell. The results showed that for the pure MB solution under white light irradiation, the absorption at 664 nm did not decrease by itself, and after adding HPS to the MB solution, the absorption of MB could not be decreased without light irradiation. It can be seen that it was the hydrogen production by HPS after light irradiation that caused the decrease in ultraviolet absorption.

[0049] Figure 5The ultraviolet absorption analysis of the MB solution with / without light was carried out by using an ultraviolet spectrophotometer for liposomes encapsulating AA (Lip with AA), Au NPs (NPs), liposomes (Lip), liposomes without photosensitizer encapsulating AA and Au NPs (Lip-A-N), lutein liposomes (Lu-Lip), lutein liposomes encapsulating Au NPs (Lu-Lip-N), lutein liposomes encapsulating AA (Lu-Lip-A) and HPS. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare a test solution, and then use an ultraviolet spectrophotometer for testing. Add deionized water and the component solutions of liposomes encapsulating AA (Comparative Example 1), Au NPs (Comparative Example 2), liposomes (Comparative Example 3), liposomes without photosensitizer encapsulating AA and Au NPs (Comparative Example 4), lutein liposomes (Comparative Example 5), lutein liposomes encapsulating Au NPs (Comparative Example 6), lutein liposomes encapsulating AA (Comparative Example 7) into the blank cell, and keep the concentration the same as that during the HPS test. Add the MB solution and the component solution with the same volume as the corresponding blank group into the sample cell, and make up the volume to 3 mL with deionized water. Detect the ultraviolet absorption peak of the sample in the range of 500 - 800 nm at an interval of 1 nm. Test once when the sample cell is just put in, and test again after irradiating the sample cell and the blank control cell with white light for 2 min simultaneously. It can be seen from the results that when each component is added to the MB solution in equivalent amounts, MB cannot decrease as much as HPS with / without light, so it can be known that a complete HPS system needs to be constructed to achieve hydrogen production.

[0050] Figure 6 The cell viability of RAW264.7 cells after adding various concentrations of HPS was tested by using a cell analyzer. Collect the cells in the logarithmic growth phase, and adjust the density of the cell suspension to 10 5 cells / mL with complete medium. Add 100 μL of the cell suspension into each well of a 96-well cell culture plate and culture it overnight in an incubator. Aspirate the medium, set up groups, add 100 μL of serum-free DMEM medium to the blank group, and add HPS solutions with concentrations of 100 / 200 / 500 / 800 / 1k / 2k / 3k / 4k / 5k μg / mL (dissolved in incomplete medium DMEM) to the HPS sample groups respectively. After culturing the 96-well plate in an incubator for 24 h, discard the old culture medium. Add 100 μL of 0.5 mg / mL MTT solution to each well in turn, and culture it in the dark for 4 h. Then carefully aspirate the supernatant, add 100 μL of DMSO to each well, and oscillate it in the dark for 3 min in an enzyme-linked immunosorbent assay (ELISA) reader to fully dissolve the purple crystal formazan, and measure the absorbance value of each well at 490 nm to calculate the cell viability. It can be seen from the results that when HPS is added to RAW246.7 cells within a certain concentration range, the cell viability is above 90%, indicating that HPS has good biocompatibility.

[0051] Figure 7 The images of MB-stained Raw264.7 cells were taken by a cell microscope under different conditions. 3 mg of methylene blue powder was dissolved in 15 mL of serum-free DMEM to prepare a staining solution. RAW264.7 cells were cultured in a 12-well plate, and different component solutions were added and incubated with the cells for 24 h (the concentration of each component was the same as that in HPS). For the HPS+L group, white light was irradiated for 10 min. After sucking out the culture medium, the cells were incubated with 1 ml of staining DMEM solution for 1 h, then the staining solution was sucked out, and the cells were washed 3 times with PBS. Subsequently, an inverted optical microscope was used to observe and take images. The results showed that HPS could fade the color of the cells stained with MB after light irradiation, indicating that HPS could achieve effective intracellular hydrogen production.

[0052] Figure 8 and Figure 9 The fluorescence of RAW264.7 cells stained with DCFH-DA and the DCF intensity of LPS-induced ROS production in RAW264.7 cells were tested by fluorescence microscope and flow cytometry respectively under different conditions. The cell precipitate in the logarithmic growth phase was collected, and the cell density was adjusted to 1×10 5 cells / mL. 2 mL of cell suspension was added to each well of a 6-well cell culture plate and cultured overnight. The experimental groups were blank group, LPS group, LPS + different concentrations (500 / 1k / 2k / 4k μg / mL) of HPS group, LPS + different concentrations (500 / 1k / 2k / 4k μg / mL) of HPS group + light irradiation group. LPS was added and incubated for 24 h. After the stimulation was completed, the culture medium was sucked out, the drug was added and incubated for 2 h. The light irradiation group was irradiated with white light for 10 min, and then sucked out. 1 mL of the probe DCFH-DA (10 μmol / mL, dissolved in DMEM) for detecting ROS was added to each well and incubated in an incubator for 20 min. After the cells were washed twice with PBS, an inverted fluorescence microscope was used to take pictures of the cells. After taking the ROS fluorescence pictures, the well plate was placed in a flow cytometer and detected under the conditions of an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The results showed that HPS effectively decreased the intracellular ROS induced by inflammation in RAW264.7 cells after adding LPS to induce inflammation and producing hydrogen by light irradiation.

[0053] Figure 10 The levels of IL-6 and THF-α in LPS-induced RAW264.7 cells were tested after different conditions of treatment. The cell precipitate in the logarithmic growth phase was collected, and the cell density was adjusted to 1×10 5cells / mL. Add 2 mL of cell suspension to each well of a 6-well cell culture plate and incubate overnight. The experimental groups are the blank group, the LPS group, the LPS + HPS groups with different concentrations (500 / 1k / 2k / 4k μg / mL), and the LPS + HPS groups with different concentrations (500 / 1k / 2k / 4k μg / mL) + light group. Add LPS and incubate for 24 h. After the stimulation is completed, aspirate the culture medium, add the drug and incubate for 2 h. Irradiate the light group with white light for 10 min. Collect the supernatant culture medium of each group and operate according to the instructions of the ELISA kit to detect the effect of HPS on the release of inflammatory factors TNF-α and IL-6 by cells. It can be seen from the results that HPS can significantly decrease the levels of intracellular inflammatory factors IL-6 and THF-α after light irradiation, indicating that HPS has a remarkable effect on the treatment of inflammation.

[0054] Figure 11 and Figure 16 The proliferation of human dermal papilla cells before and after the addition of the product was detected using a real-time cell analyzer (RTCA) (ACEA Biosciences, Inc., USA). Collect cells in the logarithmic growth phase and adjust the density of the cell suspension to 10 5 cells / mL. Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate overnight in an incubator. Aspirate the culture medium. Add 100 μL of DMEM to the blank control group, and add media containing different concentrations (100, 200, 500, 1k, 2k, 3k, 4k, and 5k μg / mL) of the hydrogen-producing liposomes HPS encapsulating Au nanoparticles obtained in Example 1 to the product groups. Set 6 replicates for each sample group at each concentration and the blank control group. After culturing the 96-well plate in the incubator for 22 h without light + 2 h with light and 24 h without light respectively, discard the old culture medium. Add 100 μL of 0.5 mg / mL MTT solution to each well in turn, incubate in the dark for 4 h, carefully aspirate the supernatant, add 100 μL of DMSO to each well, and shake in the dark in an enzyme-linked immunosorbent assay (ELISA) reader for 3 min to fully dissolve the purple crystal formazan, and measure the absorbance value of each well at 490 nm to calculate the cell proliferation rate. We found that low doses of the hydrogen-producing liposomes HPS encapsulating Au nanoparticles (100, 200, 500, 1k, 2k, 3k, and 5k μg / mL) under light irradiation can promote the proliferation ability of dermal papilla cells, and the cell proliferation rate is increased by 18% compared with the control group without adding HPS (p < 0.05), while the proliferation rate of HPS promoting dermal papilla cells without light is only 6% (p > 0.05), showing no significant difference from the blank control group. This finding indicates that HPS has a positive promoting effect on the growth and division of dermal papilla cells, and HPS can make the proliferation rate of dermal papilla cells higher after light-induced hydrogen production.

[0055] Figure 12 The ultraviolet absorption analysis of the methylene blue (MB) solution added with hydrogen-producing liposomes containing riboflavin was carried out before and after white light irradiation using an ultraviolet spectrophotometer. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare a test solution, and use an ultraviolet spectrophotometer for testing. Add deionized water and riboflavin liposome solution (comparative example 8) to the blank cell, keeping the concentration the same as that in the HPS test. Add 0.5 mL of MB solution and the same volume of component solution as the corresponding blank group to the sample cell, and make up the volume to 3 mL with deionized water. Detect the ultraviolet absorption peak of the sample in the range of 500 - 800 nm at an interval of 1 nm. Test once when the sample cell is just put in, and test respectively after irradiating the sample cell and the blank control cell with white light for 2 / 4 / 8 min simultaneously.

[0056] It can be seen from the results that for the hydrogen-producing liposomes containing riboflavin, after adding the methylene blue (MB) solution, even after white light irradiation, the absorption of MB at 664 nm did not decrease significantly, indicating that after light irradiation, the hydrogen-producing liposomes containing riboflavin produced less hydrogen.

[0057] Figure 13 The ultraviolet absorption analysis of the methylene blue (MB) solution added with hydrogen-producing liposomes containing resveratrol was carried out before and after white light irradiation using an ultraviolet spectrophotometer. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare a test solution, and use an ultraviolet spectrophotometer for testing. Add deionized water and resveratrol liposome solution (comparative example 9) to the blank cell, keeping the concentration the same as that in the HPS test. Add 0.5 mL of MB solution and the same volume of component solution as the corresponding blank group to the sample cell, and make up the volume to 3 mL with deionized water. Detect the ultraviolet absorption peak of the sample in the range of 500 - 800 nm at an interval of 1 nm. Test once when the sample cell is just put in, and test respectively after irradiating the sample cell and the blank control cell with white light for 2 / 4 / 6 / 8 / 10 min simultaneously. It can be seen from the results that for the hydrogen-producing liposomes containing resveratrol, after adding the methylene blue (MB) solution, the absorption of MB at 664 nm did not decrease significantly, but after white light irradiation (2 - 10 min), the absorption decreased significantly, indicating that after light irradiation, the hydrogen-producing liposomes containing resveratrol successfully produced hydrogen and reduced the MB in the solution. The hydrogen production effect is better than that of the hydrogen-producing liposomes containing riboflavin in comparative example 8, but not as good as that of the hydrogen-producing liposomes containing lutein (HPS).

[0058] Figure 14The ultraviolet absorption analysis of methylene blue (MB) solution added with hydrogen-producing liposomes containing sodium erythorbate was carried out by ultraviolet spectrophotometer before and after white light irradiation. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare a test solution, and use an ultraviolet spectrophotometer for testing. The blank cell is added with the product synthesized from deionized water and sodium erythorbate (comparative example 10), and the concentration is kept the same as that in the HPS test. The sample cell is added with 0.5 mL of MB solution and the same volume of component solution as the corresponding blank group, and is made up to 3 mL with deionized water. The ultraviolet absorption peak of the sample in the range of 500 - 800 nm is detected at an interval of 1 nm. The sample cell is tested once when it is just put in, and the sample cell and the blank control cell are irradiated with white light for 2 / 4 min and then tested separately. From the results, it can be seen that for the hydrogen-producing liposomes containing sodium erythorbate, after adding methylene blue (MB) solution, the absorption of MB at 664 nm did not decrease much, but after white light irradiation for 2 min, the absorption decreased significantly, indicating that after illumination, the hydrogen-producing liposomes containing sodium erythorbate successfully produced hydrogen and reduced MB in the solution. The hydrogen production effect is better than that of the hydrogen-producing liposomes containing vitamin C ethyl ether in comparative example 11, but not as good as that of the hydrogen-producing liposomes containing ascorbic acid (AA) (HPS).

[0059] Figure 15 The ultraviolet absorption analysis of methylene blue (MB) solution added with hydrogen-producing liposomes containing vitamin C ethyl ether was carried out by ultraviolet spectrophotometer before and after white light irradiation. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare a test solution, and use an ultraviolet spectrophotometer for testing. The blank cell is added with the product synthesized from deionized water and vitamin C ethyl ether (comparative example 11), and the concentration is kept the same as that in the HPS test. The sample cell is added with 0.5 mL of MB solution and the same volume of component solution as the corresponding blank group, and is made up to 3 mL with deionized water. The ultraviolet absorption peak of the sample in the range of 500 - 800 nm is detected at an interval of 1 nm. The sample cell is tested once when it is just put in, and the sample cell and the blank control cell are irradiated with white light for 2 / 4 min and then tested separately. From the results, it can be seen that for the hydrogen-producing liposomes containing vitamin C ethyl ether, after adding methylene blue (MB) solution, the absorption of MB at 664 nm did not decrease even after white light irradiation, indicating that after illumination, the hydrogen-producing liposomes containing vitamin C ethyl ether did not produce hydrogen. This may be related to the fact that vitamin C ethyl ether is obtained by ethyl etherification modification of ascorbic acid, and its 3-position hydroxyl group is replaced by an ethyl group. This modification reduces the electron-donating ability of the hydroxyl group in the molecule, and the weakening of the electron-donating ability.

[0060] Figure 17 The staining of human dermal papilla cells under different conditions was tested by microscope. Take the cells in the logarithmic growth phase, digest, count them, and then use 1.5×10 5Cells were seeded in 6-well plates at a density of 1.5×10 cells / mL. The experiments were divided into a blank group (Control), a model group (Model, 100 ng / mL dihydrotestosterone), an HPS group (100 ng / mL dihydrotestosterone + 500 μg / mL HPS), and an HPS+L group (100 ng / mL dihydrotestosterone + 500 μg / mL HPS + light). After 24 h of drug incubation, the culture medium was aspirated, and the cells were washed twice with PBS buffer. Then, 1 mL of cell fixative was added and the cells were fixed at room temperature for 15 min and then aspirated. The cells were washed three times with PBS buffer, and 1 mL of β-galactosidase staining working solution was added. The plates were sealed with parafilm and incubated overnight at 37 °C. The cell staining was observed and photographed under an inverted microscope. The results showed that dihydrotestosterone could induce the senescence of dermal papilla cells, while hydrogen-producing liposomes HPS (500 μg / mL) encapsulated with Au nanoparticles could inhibit the activity of β-galactosidase and delay cell senescence under light illumination.

[0061] Figure 18 The fluorescence of human dermal papilla cells under different conditions after DCFH-DA staining and the DCF intensity of ROS generated by H2O2 (100 μM) in human dermal papilla cells were measured using a fluorescence microscope and a flow cytometer, respectively. Cells in the logarithmic growth phase were digested, counted, and seeded in 6-well plates at a density of 1.5×10 5 cells / mL. The experiments were divided into a blank group (Control), a model group (Model, 100 μM H2O2), an HPS group (100 μM H2O2 + 500 μg / mL HPS), and an HPS+L group (100 μM H2O2 + 500 μg / mL HPS + light). According to the instructions of the reactive oxygen species kit, DCFH-DA was diluted to 10 μM at a ratio of 1:1000. After 4 h of drug incubation, the culture medium was removed, and 1 mL of the diluted DCFH-DA solution was added to each well and incubated in an incubator for 20 min. Then, the cells were taken out and washed three times with serum-free DMEM medium. Photographs were taken under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). The cells were digested with trypsin, collected, the cell suspension was counted, and seeded in black-edged 96-well plates at a density of 5×10 4 cells / mL for real-time fluorescence detection. The results showed that in human dermal papilla cells induced by H2O2 (100 μM) with inflammation, after adding HPS and hydrogen production under light illumination, the intracellular ROS caused by inflammation decreased significantly.

[0062] Figure 19 The levels of IL-6 and IL-1α in human dermal papilla cells induced by dihydrotestosterone (0.2 μg / mL) under different treatment conditions were measured. The dermal papilla cell suspension was seeded at a density of 1×10 5Cells were seeded into 6-well plates at a density of

[0063] The results showed that compared with the Control group, the contents of inflammatory factors IL-6 and IL-1α in dermal papilla cells increased significantly after adding dihydrotestosterone (0.2 μg / mL). Under light illumination, HPS could significantly inhibit the expression of inflammatory factors.

[0064] In summary, the present invention provides a construction of a hydrogen-producing system (HPS, Hydrogen Producing System) based on Au and Pt nanoparticles. In the present invention, Au and Pt nanoparticles with high dispersibility and small size were successfully prepared based on the sodium borohydride reduction method. Liposomes with a size of 2-3 μm were prepared by the thin film hydration method to encapsulate Au and Pt nanoparticles and antioxidants, and a tightly bound hydrogen-producing nanoreactor was successfully constructed, which could produce hydrogen under natural white light illumination. Liposomes provide a unique reactor design. By keeping hydrophilic reactants (antioxidants and Au / Pt NPs) in their aqueous core and amphiphilic reactants (bioactive compounds) in the lipid bilayer, reaction molecules can be uniquely confined within the nanoscale. The liposome assembly can establish an optimal reaction environment for the reaction molecules under study.

[0065] By adjusting the ratio of liposome raw materials and encapsulating antioxidants and Au / Pt NPs through ice bath hydration, a stable hydrogen-producing system based on Au and Pt nanoparticles was synthesized, which could effectively produce hydrogen after natural light illumination and rapidly fade methylene blue. In addition, the developed light-driven system effectively reduced oxidative stress, revealing its great potential for alleviating tissue inflammation.

[0066] Comparative Example 1: Liposomes encapsulating AA (Lip with AA)

[0067] 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, and 18 mg of polyethylene oxide polymer (average molecular weight 2000) were dissolved in 60 mL of ethanol and placed in a rotary evaporator. At 45 °C, the organic solvent was removed. The obtained film was dissolved in 100 ml of deionized water, and deionized water containing 6 g of L-ascorbic acid was added. Liposomes encapsulating ascorbic acid (AA) were obtained.

[0068] Comparative Example 2: Au NPs (NPs)

[0069] HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath. Deionized water was added to the flask and stirred rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. 0.5 mL of the aqueous chloroauric acid solution was taken, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and the mixture was stirred for 20 minutes. 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL was gradually added dropwise. After 1 h, the liquid was placed in a 14 kDa MW dialysis bag and dialyzed with deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, which was stored at 4 °C in the refrigerator for later use.

[0070] Comparative Example 3: Liposome (Lip)

[0071] 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, and 18 mg of polyoxyethylene polymer (average molecular weight 2000) were dissolved in 60 mL of ethanol and placed in a rotary evaporator. At 45 °C, the organic solvent was removed, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0072] Comparative Example 4: Liposome without photosensitizer encapsulating AA and Au NPs (Lip-A-N)

[0073] HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath. Deionized water was added to the flask and stirred rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. 0.5 mL of the aqueous chloroauric acid solution was taken, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and the mixture was stirred for 20 minutes. 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL was gradually added dropwise. After 1 h, the liquid was placed in a 14 kDa MW dialysis bag and dialyzed with deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, which was stored at 4 °C.

[0074] 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, and 18 mg of polyoxyethylene polymer (average molecular weight 2000) were dissolved in 60 mL of ethanol and placed in a rotary evaporator. At 45 °C, the organic solvent was removed, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0075] 10 mL of the aqueous solution of Au nanoparticles obtained in the above step was mixed with 10 mL of liposomes, 1.2 g / L of L-ascorbic acid was added, and the mixture was ultrasonically hydrated in an ice bath for 30 min to obtain liposomes without photosensitizer encapsulating AA and Au NPs.

[0076] Comparative Example 5: Lutein liposome (Lu-Lip)

[0077] Dissolve 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyethylene oxide polymer (average molecular weight 2000), and 2 mg of lutein in 60 mL of ethanol, and place it in a rotary evaporator. Remove the organic solvent at 45 °C. Dissolve the obtained film in 100 mL of deionized water to obtain lutein liposomes.

[0078] Comparative Example 6: Lutein liposomes encapsulating Au NPs (Lu-Lip-N)

[0079] Add HAuCl4·4H2O to a round-bottom flask in an ice-water bath. Add deionized water to the flask and stir rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. Take 0.5 mL of the aqueous solution of chloroauric acid, add 10 mL of water, add 50 mg of Tween 80 as a stabilizer, stir for 20 minutes, and gradually add 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL. After 1 h, place the liquid in a 14 kDa MW dialysis bag and dialyze with deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, and store it in a refrigerator at 4 °C.

[0080] Dissolve 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyethylene oxide polymer (average molecular weight 2000), and 2 mg of lutein in 60 mL of ethanol, and place it in a rotary evaporator. Remove the organic solvent at 45 °C. Dissolve the obtained film in 100 mL of deionized water to obtain liposomes.

[0081] Mix 10 mL of the aqueous solution of Au nanoparticles obtained in the above step with 10 mL of liposomes, and ultrasonically hydrate in an ice bath for 30 min to obtain lutein liposomes encapsulating Au NPs.

[0082] Comparative Example 7: Lutein liposomes encapsulating AA (Lu-Lip-A)

[0083] Dissolve 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyethylene oxide polymer (average molecular weight 2000), and 2 mg of lutein in 60 mL of ethanol, and place it in a rotary evaporator. Remove the organic solvent at 45 °C. Dissolve the obtained film in 100 mL of deionized water to obtain liposomes.

[0084] Add 10 mL of the liposomes obtained in the above step to 0.6 g of ascorbic acid and ultrasonically hydrate in an ice bath for 30 min to obtain lutein liposomes encapsulating AA.

[0085] Comparative Example 8:

[0086] Step 1, Preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath. Deionized water was added to the flask and stirred rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. 0.5 mL of the chloroauric acid aqueous solution was taken, 10 mL of water was added, and 50 mg of Tween 80 was added as a stabilizer. After stirring for 20 minutes, 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL was gradually added dropwise. After 1 h, the liquid was placed in a 14 kDa MW dialysis bag and dialyzed with deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, which was stored at 4 °C in the refrigerator.

[0087] Step 2, Preparation of riboflavin-containing liposomes: 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average molecular mass 2000), and 2 mg of riboflavin were dissolved in 60 mL of ethanol and placed in a rotary evaporator. At 45 °C, the organic solvent was removed, and the resulting film was dissolved in 100 mL of deionized water to obtain liposomes.

[0088] Step 3, Preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of the liposomes obtained in Step 2, 1.2 g of L-ascorbic acid, and 10 mL of the aqueous solution of Au nanoparticles obtained in Step 1 were ultrasonically hydrated in an ice bath for 30 min to obtain hydrogen-producing liposomes encapsulating Au nanoparticles.

[0089] Comparative Example 9:

[0090] Step 1, Preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath. Deionized water was added to the flask and stirred rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. 0.5 mL of the chloroauric acid aqueous solution was taken, 10 mL of water was added, and 50 mg of Tween 80 was added as a stabilizer. After stirring for 20 minutes, 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL was gradually added dropwise. After 1 h, the liquid was placed in a 14 kDa MW dialysis bag and dialyzed with deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, which was stored at 4 °C in the refrigerator for later use.

[0091] Step 2, Preparation of resveratrol-containing liposomes: 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average molecular mass 2000), and 2 mg of resveratrol were dissolved in 60 mL of ethanol and placed in a rotary evaporator. At 45 °C, the organic solvent was removed, and the resulting film was dissolved in 100 mL of deionized water to obtain liposomes.

[0092] Step 3, Preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of the liposomes obtained in Step 2, 1.2 g of ascorbic acid, and 10 mL of the Au nanoparticle aqueous solution obtained in Step 1 were ultrasonically hydrated in an ice bath for 30 min to obtain hydrogen-producing liposomes encapsulating Au nanoparticles.

[0093] Comparative Example 10:

[0094] Step 1, Preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath. Deionized water was added to the flask and stirred rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. 0.5 mL of the chloroauric acid aqueous solution was taken, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and the mixture was stirred for 20 minutes. 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL was gradually added dropwise. After 1 h, the liquid was placed in a 14 kDa MW dialysis bag and dialyzed against deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, which was stored at 4 °C in a refrigerator for later use.

[0095] Step 2, Preparation of liposomes containing lutein: 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average molecular mass 2000), and 2 mg of lutein were dissolved in 60 mL of ethanol and placed in a rotary evaporator. At 45 °C, the organic solvent was removed, and the resulting film was dissolved in 100 mL of deionized water to obtain liposomes.

[0096] Step 3, Preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of the liposomes obtained in Step 2, 1.2 g of sodium erythorbate, and 10 mL of the Au nanoparticle aqueous solution obtained in Step 1 were ultrasonically hydrated in an ice bath for 30 min to obtain hydrogen-producing liposomes encapsulating Au nanoparticles.

[0097] Comparative Example 11:

[0098] Step 1, Preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath. Deionized water was added to the flask and stirred rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. 0.5 mL of the chloroauric acid aqueous solution was taken, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and the mixture was stirred for 20 minutes. 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL was gradually added dropwise. After 1 h, the liquid was placed in a 14 kDa MW dialysis bag and dialyzed against deionized water for 24 h to prepare an aqueous solution of Au nanoparticles, which was stored at 4 °C.

[0099] Step 2, Preparation of lutein liposomes: Dissolve 93 mg of dipalmitoyl phosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyethylene oxide polymer (average molecular weight 2000), and 2 mg of lutein in 60 mL of ethanol, and place it in a rotary evaporator. Remove the organic solvent at 45 °C, and dissolve the obtained film in 100 mL of deionized water to obtain liposomes.

[0100] Step 3, Preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: Ultrasonically hydrate 10 mL of the liposomes obtained in Step 2, 1.2 g of vitamin C ethyl ether, and 10 mL of the aqueous Au nanoparticle solution obtained in Step 1 in an ice bath for 30 min to obtain hydrogen-producing liposomes encapsulating Au nanoparticles.

[0101] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Use of a hydrogen-producing liposome in the preparation of a product for hydrogen production, anti-inflammation, antioxidant and anti-hair loss, characterized in that: The preparation method of the hydrogen-producing liposome is as follows: Prepare a nanoparticle solution: Add a gold salt to a solvent, add a stabilizer and a reducing agent for reaction, and obtain the nanoparticle solution after dialysis; Prepare a liposome containing a bioactive compound; the bioactive compound is lutein; Prepare a hydrogen-producing liposome: Mix the liposome containing lutein, an antioxidant and the nanoparticle solution and perform ultrasonic treatment to obtain the hydrogen-producing liposome; the antioxidant is ascorbic acid; The preparation of the nanoparticle solution is to add HAuCl4·4H2O to a round-bottom flask in an ice-water bath, add deionized water to the flask and stir rapidly to prepare an aqueous solution of chloroauric acid with a concentration of 0.1 mM. Take 0.5 mL of the aqueous solution of chloroauric acid, add 10 mL of water, add 50 mg of Tween 80 as a stabilizer, stir for 20 minutes, and gradually add 2.5 mL of a NaBH4 reducing agent with a concentration of 2.4 mg / mL. After 1 h, put the obtained liquid into a 14 kDa MW dialysis bag and dialyze with deionized water for 24 h to obtain the nanoparticle solution; Among them, the preparation of the liposome containing a bioactive compound is to dissolve dipalmitoyl phosphatidylcholine, cholesterol, Tween 80, a polyethylene oxide polymer and lutein in a solvent, remove the solvent, and dissolve the product in deionized water to obtain a liposome with a concentration of 0.02-2 wt%; the concentration of lutein is 0.0001~0.005%; the mass ratio of dipalmitoyl phosphatidylcholine, cholesterol, Tween 80, and polyethylene oxide polymer is 15~25:4~5:0.3~5:3~8; For the preparation of the hydrogen-producing liposome, the mass ratio of the liposome containing lutein to the nanoparticle solution is 1:0.5~2; the concentration of the antioxidant is 0.5~20 wt%.

2. The application according to claim 1, wherein: The removal of the solvent includes rotary evaporation to remove the solvent at a temperature of 45°C.

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