Preparation method of hydrogen-producing lipidosome, hydrogen-producing lipidosome and application of hydrogen-producing lipidosome in hydrogen-producing, anti-inflammatory and anti-alopecia products

Abstract

The invention discloses a preparation method of hydrogen-producing lipidosome, the hydrogen-producing lipidosome and application of the hydrogen-producing lipidosome in hydrogen-producing, anti-inflammatory and anti-alopecia products, the stable and dense lipidosome is prepared through a simple hydration method, the reaction time is only 15 min, the needed temperature is only 45-50 DEG C, the synthesized lipidosome is good in dispersity and high in stability, and the prepared lipidosome can be used for preparing the anti-inflammatory and anti-alopecia products. The wrapping property is good. The preparation method is simple in process, mild in reaction condition, short in reaction time, low in energy consumption and capable of realizing large-scale production. And in the subsequent antioxidant and Au / Pt / Ag NPs wrapping reaction, the synthesized lipidosome and the wrapping substance are mixed and subjected to ice bath ultrasonic treatment, and the assembled hydrogen production system can be obtained. The reaction conditions are simple, convenient and rapid, and the success rate is high, so that a new opportunity is developed for a hydrogen production system based on Au, Pt and Ag nanoparticles in the aspects of biomedical treatment and cosmetic anti-inflammation 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 and hydrogen-producing liposomes and applications of the hydrogen-producing liposomes in hydrogen-producing, anti-inflammatory and anti-hair loss products. Background Art

[0002] Hydrogen (H2) is a potential antioxidant that 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 through 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, and it is difficult to maintain a high concentration for a long time in the area that needs to be treated.

[0003] The structure of liposomes is that the outside is surrounded by hydrophilic heads and the inside is connected in the form of a phospholipid bilayer sphere with a hydrophobic tail. 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, the use of lipids as a combination material makes liposomes biocompatible and can be used in a variety of applications such as cosmetics, medicine, and drug delivery.

[0004] Metal nanoparticles have been widely used in various fields due to their excellent physical and chemical properties. Summary of the invention

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

[0006] As one aspect of the present invention, the present invention provides a method for preparing hydrogen-producing liposomes, which comprises: Preparation of nanoparticle solution: adding gold salt, platinum salt or silver salt to a solvent, adding a stabilizer and a reducing agent to react, and obtaining a nanoparticle solution after dialysis; Preparing liposomes containing bioactive compounds; the bioactive compounds include one or more of lutein, vitamin E, coumarin, carotenoids, resveratrol, lycopene, riboflavin, curcumin, and rheum; Preparation of hydrogen-producing liposomes: mixing the liposomes containing the bioactive compound, the antioxidant and the nanoparticle solution and then ultrasonically treating them to obtain hydrogen-producing liposomes; the antioxidant comprises 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 and sodium sulfite.

[0007] As a preferred embodiment of the method for preparing 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.

[0008] As a preferred embodiment of the method for preparing hydrogen-producing liposomes of the present invention, the solvent includes water, the reaction temperature is 0-8° C., and the reaction time is 0.5-2 h.

[0009] As a preferred embodiment of the method for preparing the hydrogen-producing liposomes of the present invention: the stabilizer includes Tween 80, and the concentration of Tween 80 is 0.01-1wt%; the reducing agent includes sodium borohydride, and 20% of 1-10 mg / mL sodium borohydride is added; the dialysis time is 6-24 h.

[0010] As a preferred embodiment of the method for preparing the hydrogen-producing liposomes described in the present invention, the liposomes include several of dipalmitoylphosphatidylcholine, cholesterol, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine, Tween, sodium deoxycholate, polyethylene glycol 2000, octadecylamine, sodium taurocholate, polyoxyethylene polymers, dioleoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine polyethylene glycol 2000.

[0011] As a preferred embodiment of the method for preparing the hydrogen-producing liposomes of the present invention, dipalmitoylphosphatidylcholine, cholesterol, Tween, polyoxyethylene polymer and biologically active compounds 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 0.02-2wt%; wherein the concentration of the biologically active compound is 0.0001-0.005%; wherein the mass ratio of dipalmitoylphosphatidylcholine, cholesterol, Tween and polyoxyethylene polymer is 15-25:4-5:0.3-5:3-8.

[0012] As a preferred embodiment of the method for preparing hydrogen-producing liposomes of the present invention: the solvent removal comprises rotary evaporation to remove the solvent at a temperature of 45°C.

[0013] As a preferred embodiment of the method for preparing 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; and the concentration of the antioxidant is 0.5-20wt%.

[0014] Beneficial effects of the present invention: The present invention constructs a hydrogen production system in liposomes, providing unprecedented insights into 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 tightly fitting hydrogen production nanoreactor, which can effectively reduce the ultraviolet absorption peak of methylene blue at 664nm under natural white light irradiation, creating a new hydrogen production system.

[0015] The method described in the present invention prepares stable and dense liposomes by a simple hydration method, and the reaction time is only 15min, and the required temperature is only 45-50°C. The synthesized liposomes have good dispersibility, strong stability, and good encapsulation. The preparation method is simple in process, mild in reaction conditions, short in reaction time, low in 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 encapsulated materials in an ice bath ultrasound to obtain an assembled hydrogen production system. The reaction conditions are simple, fast, and have a high success rate, thereby opening up new opportunities for hydrogen production systems based on Au, Pt, and Ag nanoparticles in biomedicine and cosmetics anti-inflammatory and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein: Figure 1 It is a transmission electron microscope image and a particle size analysis image of the particle size distribution in the example of the present invention.

[0017] Figure 2 This is an image of the synthetic liposome in the example of the present invention observed under a microscope.

[0018] Figure 3 This is the UV image of methylene blue MB solution added with HPS before and after white light irradiation.

[0019] Figure 4 These are the UV images of pure methylene blue MB under white light (upper image) and the UV image of methylene blue MB solution added to HPS without light within 10 minutes (lower image).

[0020] Figure 5 The UV absorption graphs of MB solution with and without light added to AA-encapsulated liposomes, Au NPs, liposomes and HPS (upper graph) and the UV absorption graphs of MB solution with and without light added to photosensitizer-free liposomes encapsulated AA and Au NPs, lutein liposomes, lutein liposomes encapsulated Au NPs, lutein liposomes encapsulated AA and HPS (lower graph).

[0021] Figure 6 is the survival rate of RAW264.7 cells after adding various concentrations of HPS.

[0022] Figure 7 Microscope images of Raw264.7 cells stained with MB under different conditions: (a) no treatment, (b) with the addition of HPS, and (c) with illumination after the addition of HPS.

[0023] Figure 8 Fluorescence microscopy images of Raw264.7 cells after DCFH-DA staining under different conditions.

[0024] Fig. 9 DCF intensity of ROS produced by LPS-induced RAW264.7 cells after different treatments.

[0025] Fig.10 (Left) shows the IL-6 level in RAW264.7 cells induced by lps after different treatments. (Right) shows the THF-α level in RAW264.7 cells induced by lps after different treatments.

[0026] Fig.11 This is the cell proliferation of human hair papilla dermal cells after adding various concentrations of HPS and irradiating with light.

[0027] Fig.12 This is the UV image of MB solution added to hydrogen-producing liposomes containing riboflavin before and after white light irradiation.

[0028] Fig.13 This is the UV image of MB solution added to hydrogen-producing liposomes containing resveratrol before and after white light irradiation.

[0029] Fig.14 This is the UV image of MB solution added to hydrogen-producing liposomes containing sodium isoascorbate before and after white light irradiation.

[0030] Fig.15 This is the UV image of MB solution added to hydrogen-producing liposomes containing vitamin C ethyl ether before and after white light irradiation.

[0031] Fig.16 This is the cell proliferation of human dermal papilla cells after adding various concentrations of HPS without light.

[0032] Fig.17 This is an optical microscopic image of human dermal papilla cells stained with β-galactosidase.

[0033] Fig.18 Fluorescence microscopy images of human dermal papilla cells after DCFH-DA staining before and after illumination (left) and the DCF intensity of ROS produced by human dermal papilla cells induced by H2O2 (right).

[0034] Fig.19The levels of IL-1α (left) and IL-6 (right) in human dermal papilla cells induced by dihydrotestosterone before and after light exposure. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0036] Embodiment 1: Step 1, preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k Da MW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C.

[0037] Step 2, preparation of lutein-containing liposomes: 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween 80, 18 mg of polyoxyethylene polymer (average molecular weight 2000, CAS number: 25322-68-3) and 2 mg of lutein were mixed and dissolved in 60 mL of ethanol, and placed in a rotary evaporator at 45° C. to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0038] Step 3, preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of liposomes obtained in step 2 were ultrasonically hydrated in an ice bath for 30 min with 1.2 g L-ascorbic acid and 10 mL of the Au nanoparticle aqueous solution obtained in step 1 to obtain hydrogen-producing liposomes encapsulating Au nanoparticles (hereinafter referred to as HPS).

[0039] Figure 1 TEM transmission electron microscopy was used to analyze the particle size of AuNPs. The Au nanoparticle suspension was deposited on a 400-mesh carbon-film copper grid and the TEM image was taken. About 50 individual particles were counted from the TEM image taken, and the average diameter (Dc) of each AuNPs sample was calculated. The results show that the synthesized Au NPs have a particle size of 2-3nm and are evenly and densely dispersed.

[0040] Figure 2The hydrogen-producing liposomes (hereinafter referred to as HPS) encapsulating Au nanoparticles were observed under a microscope. The liquid to be observed was smeared on a glass sheet, placed under a hot stage polarizing microscope, and the HPS was observed and imaged at 10*50 times. The results show that the HPS is evenly dispersed under the microscope, and the vesicles are dense and uniform in size.

[0041] Figure 3 The ultraviolet absorption analysis of the methylene blue (MB) solution before and after the addition of HPS to the white light irradiation was performed using an ultraviolet spectrophotometer. 18.6 mg of methylene blue powder was weighed and dissolved in 50 mL of deionized water to form a test solution, and the test was performed using an ultraviolet spectrophotometer. 2 mL of deionized water and 1 mL of HPS solution were added to the blank pool, and 1.5 mL of deionized water, MB solution and 1 mL of HPS solution were added to the sample pool. The ultraviolet absorption peak of the sample in the 500-800 nm range was detected at intervals of 1 nm. The sample pool was tested once when it was just put in, and the sample pool and the blank control pool were tested once after being irradiated with white light for 2 minutes. It can be seen from the results that the hydrogen-producing liposomes (HPS) synthesized in step 3 of the embodiment did not reduce the absorption of MB at 664 nm much after the addition of methylene blue (MB) solution (HPS group), but after white light irradiation (HPS+L group), the absorption dropped sharply, indicating that after illumination, HPS successfully produced hydrogen and reduced MB in the solution. Figure 3 In the figure, the HPS group is the light absorption at 664nm when HPS and methylene blue MB are added without light, the MB group is the light absorption at 664nm when methylene blue is added, and the HPS+L group is the light absorption at 664nm when HPS and methylene blue MB are added with light.

[0042] Figure 4 The UV absorption of pure methylene blue MB under white light irradiation (upper figure) and methylene blue MB solution added to HPS without light (lower figure) was analyzed by UV spectrophotometer within 10 minutes.

[0043] Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare the test solution. Use a UV spectrophotometer to test. Add deionized water to the blank cell and MB solution to the sample cell. Detect the UV absorption peak of the sample in the 500-800 nm range at intervals of 1 nm. Test once when the sample cell is just put in, and test the sample cell separately after irradiating it with white light for different periods of time. From the results, it can be seen that the absorption of pure MB solution at 664 nm will not decrease by itself under white light irradiation, and after HPS is added to the MB solution, the MB absorption cannot be reduced without light irradiation. It can be seen that HPS produces hydrogen after light irradiation, which causes the UV absorption to decrease.

[0044] Figure 5The UV absorption of MB solution with and without light was analyzed by UV spectrophotometer with the addition of AA liposomes (Lip with AA), Au NPs (NPs), liposomes (Lip), liposomes without photosensitizer containing AA and Au NPs (Lip-AN), lutein liposomes (Lu-Lip), lutein liposomes containing Au NPs (Lu-Lip-N), lutein liposomes containing 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 the test solution. Use a UV spectrophotometer to test. Add deionized water and AA-encapsulated liposomes (Comparative Example 1), Au NPs (Comparative Example 2), liposomes (Comparative Example 3), AA and Au NPs-encapsulated liposomes without photosensitizer (Comparative Example 4), lutein liposomes (Comparative Example 5), AuNPs-encapsulated lutein liposomes (Comparative Example 6), and AA-encapsulated lutein liposomes (Comparative Example 7) to the blank pool. Keep the concentration consistent with that in the HPS test. Add MB solution and the same volume of component solution of the corresponding blank group to the sample pool, and dilute to 3 mL with deionized water. Detect the ultraviolet absorption peak of the sample in the 500-800 nm range at an interval of 1 nm. Test once when the sample pool is just put in, and test once after 2 minutes of white light irradiation for the sample pool and the blank control pool. From the results, it can be seen that when each component is added to the MB solution in equivalent amounts, the MB cannot be reduced as much as the HPS in the presence or absence of light. This shows that a complete HPS system must be constructed to achieve hydrogen production.

[0045] Figure 6 The cell viability of RAW264.7 cells after adding HPS at different concentrations was tested using a cell analyzer. Cells in the logarithmic growth phase were collected and the cell suspension density was adjusted to 10 5 / mL, add 100 μL of cell suspension to each well of a 96-well cell culture plate, and culture overnight in an incubator. Aspirate the culture medium, set up groups, add 100 μL of serum-free DMEM culture medium to the blank group, add 100 / 200 / 500 / 800 / 1k / 2k / 3k / 4k / 5k μg / mL HPS solution (dissolved in incomplete culture medium DMEM) to the HPS sample group, place 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, culture in the dark for 4 h, carefully aspirate the supernatant, add 100 μL DMSO to each well, shake in the dark for 3 min in an ELISA reader to fully dissolve the purple crystalline diformazan, and measure the absorbance value of each well at 490 nm to calculate the cell survival rate. The results show that when HPS is added to RAW246.7 cells within a certain concentration, the cell survival rate is above 90%, indicating that HPS has good biocompatibility.

[0046] Figure 7 Images of Raw264.7 cells stained with MB were taken under different conditions using a cell microscope. 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 on a 12-well plate, and different component solutions were added to the cells for 24 hours (the concentration of the single component was consistent with that in HPS). The HPS+L group was irradiated with white light for 10 minutes, the culture medium was aspirated, and 1 ml of the dyed DMEM solution was incubated for 1 hour. The staining solution was aspirated, washed 3 times with PBS, and then observed and photographed using an inverted optical microscope. The results show that HPS can fade the color of cells stained with MB after illumination, indicating that HPS can achieve effective intracellular hydrogen production.

[0047] Figure 8 and Fig. 9 Fluorescence microscopy and flow cytometry were used to test the fluorescence of RAW264.7 cells after DCFH-DA staining under different conditions and the DCF intensity of ROS produced by LPS-induced RAW264.7 cells. Cell pellets in the logarithmic growth phase were collected and the cell density was adjusted to 1×10 5 / 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) HPS group, LPS + different concentrations (500 / 1k / 2k / 4k μg / mL) HPS group + light group, and LPS was added to stimulate for 24 h. After the stimulation, the culture medium was aspirated, the drug was added and incubated for 2 h, the light group was irradiated with white light for 10 min, then aspirated, and 1 mL of ROS detection probe DCFH-DA (10 μmol / mL, dissolved in DMEM) was added to each well and incubated in the incubator for 20 min. After washing the cells twice with PBS, the cells were photographed using an inverted fluorescence microscope. After taking the ROS fluorescence picture, the well plate was placed in a flow cytometer and detected under the conditions of 488 nm excitation wavelength and 525 nm emission wavelength. The results show that HPS can effectively reduce the intracellular ROS caused by inflammation after being added to LPS-induced inflammatory RAW264.7 cells and producing hydrogen under light.

[0048] Fig.10 The levels of IL-6 and THF-α induced by lps in RAW264.7 cells were tested after different treatment conditions. Cell pellets in the logarithmic growth phase were collected and the cell density was adjusted to 1×10 5 / 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) HPS group, LPS + different concentrations (500 / 1k / 2k / 4k μg / mL) HPS group + light group, and LPS was added to stimulate for 24 h. After the stimulation, the culture medium was aspirated, the drug was added and incubated for 2h, the light group was irradiated with white light for 10min, the supernatant culture medium of each group was collected, and the ELISA kit instructions were followed 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 reduce the levels of intracellular inflammatory factors IL-6 and THF-α after light exposure, which shows that HPS has a considerable effect on the treatment of inflammation.

[0049] Fig.11 and Fig.16 A real-time cell analyzer (RTCA) (ACEA Biosciences, USA) was used to detect the proliferation of human hair papilla dermal cells before and after the addition of the product. Cells in the logarithmic growth phase were collected and the cell suspension density was adjusted to 10 5 / mL, add 100 μL of cell suspension to each well of a 96-well cell culture plate and culture overnight in an incubator. Aspirate the culture medium, remove the culture medium, add 100 μL DMEM to the blank control group, and add culture medium containing different concentrations of (100, 200, 500, 1k, 2k, 3k, 4k and 5k μg / mL) of hydrogen-producing liposomes HPS encapsulating Au nanoparticles obtained in Example 1 to the product group. Six replicate wells are set for each concentration of the sample group and the blank control group. The 96-well plate was placed in an incubator for 22 h of incubation without light + 2 h of incubation with light and 24 h of incubation without light, then the old culture medium was discarded, 100 μL of 0.5 mg / mL MTT solution was added to each well in turn, and after 4 h of incubation in the dark, the supernatant was carefully aspirated, 100 μL of DMSO was added to each well, and the plate was shaken in the dark for 3 min in an ELISA reader to fully dissolve the purple crystalline diformazan, and the absorbance value of each well at 490 nm was measured to calculate the cell proliferation rate. We found that low doses of hydrogen-producing liposomes HPS (100, 200, 500, 1k, 2k, 3k and 5k μg / mL) encapsulating Au nanoparticles under light can promote the proliferation of hair papilla cells, and the cell proliferation rate was increased by 18% compared with the control group without HPS (p<0.05), while the proliferation rate of hair papilla cells promoted by HPS in the dark was only 6% (p>0.05), which was not significantly different from the blank control group. This finding suggests that HPS has a positive effect on the growth and division of hair papilla cells, and HPS can promote the proliferation rate of hair papilla cells to a higher level after producing hydrogen under light.

[0050] Fig.12 The ultraviolet absorption analysis of the methylene blue MB solution before and after white light irradiation was performed by ultraviolet spectrophotometer. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare the test solution. Use ultraviolet spectrophotometer for testing. Add deionized water and riboflavin liposome solution to the blank pool (Comparative Example 8), keep the concentration consistent with that in the HPS test, add 0.5 mL of MB solution and the same volume of component solution of the corresponding blank group to the sample pool, and dilute to 3 mL with deionized water. Detect the ultraviolet absorption peak of the sample in the 500-800 nm range at intervals of 1 nm. Test once when the sample pool is just put in, and test the sample pool and the blank control pool separately after 2 / 4 / 8 minutes of white light irradiation.

[0051] From the results, it can be seen that after adding methylene blue (MB) solution to the hydrogen-producing liposomes containing riboflavin, the absorption of MB at 664nm does not decrease much even after white light irradiation, indicating that after light irradiation, the hydrogen-producing liposomes containing riboflavin produce less hydrogen.

[0052] Fig.13 The ultraviolet absorption analysis of the methylene blue MB solution with hydrogen-producing liposomes containing resveratrol before and after white light irradiation was performed using an ultraviolet spectrophotometer. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare the test solution. Use an ultraviolet spectrophotometer for testing. Add deionized water and resveratrol liposome solution (Comparative Example 9) to the blank pool, keep the concentration consistent with that in the HPS test, add 0.5 mL of MB solution and the same volume of component solution of the corresponding blank group to the sample pool, and dilute to 3 mL with deionized water. Detect the ultraviolet absorption peak of the sample in the 500-800 nm range at intervals of 1 nm. Test once when the sample pool is just put in, and test the sample pool and the blank control pool separately after irradiating with white light for 2 / 4 / 6 / 8 / 10 minutes. The results show that the hydrogen-producing liposomes containing resveratrol did not reduce the absorption of MB at 664nm after adding methylene blue (MB) solution, but the absorption decreased significantly after white light irradiation (2-10min), indicating that after light irradiation, the hydrogen-producing liposomes containing resveratrol successfully produced hydrogen and reduced MB in the solution. The hydrogen production effect is worse than that of the hydrogen-producing liposomes containing riboflavin in comparative example 8, but not as good as the hydrogen-producing liposomes (HPS) containing lutein.

[0053] Fig.14The ultraviolet absorption analysis of the methylene blue MB solution with hydrogen-producing liposomes containing sodium isoascorbate before and after white light irradiation was performed using an ultraviolet spectrophotometer. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare the test solution, and test it using an ultraviolet spectrophotometer. Add deionized water and the product synthesized by sodium isoascorbate (Comparative Example 10) to the blank pool, keeping the concentration consistent with that in the HPS test, add 0.5 mL of MB solution and the same volume of component solution of the corresponding blank group to the sample pool, and dilute to 3 mL with deionized water, and detect the ultraviolet absorption peak of the sample in the 500-800 nm range at intervals of 1 nm. Test once when the sample pool is just put in, and test the sample pool and the blank control pool separately after irradiating with white light for 2 / 4 minutes. The results show that the hydrogen-producing liposomes containing sodium isoascorbate did not reduce the absorption of MB at 664nm after adding methylene blue (MB) solution, but the absorption decreased significantly after 2 minutes of white light irradiation, indicating that after light irradiation, the hydrogen-producing liposomes containing sodium isoascorbate successfully produced hydrogen and reduced MB in the solution. The hydrogen production effect is worse than that of the hydrogen-producing liposomes containing vitamin C ethyl ether in Comparative Example 11, but not as good as the hydrogen-producing liposomes (HPS) containing ascorbic acid (AA).

[0054] Fig.15 The ultraviolet absorption analysis of the methylene blue MB solution with hydrogen-producing liposomes containing vitamin C ethyl ether before and after white light irradiation was performed using an ultraviolet spectrophotometer. Weigh 18.6 mg of methylene blue powder and dissolve it in 50 mL of deionized water to prepare the test solution, and use an ultraviolet spectrophotometer for testing. Add deionized water and the product synthesized by vitamin C ethyl ether to the blank pool (Comparative Example 11), keep the concentration consistent with that in the HPS test, add 0.5 mL of MB solution and the same volume of component solution of the corresponding blank group to the sample pool, and dilute to 3 mL with deionized water, and detect the ultraviolet absorption peak of the sample in the 500-800 nm range at intervals of 1 nm. Test once when the sample pool is just put in, and test the sample pool and the blank control pool separately after 2 / 4 minutes of white light irradiation. The results show that after adding methylene blue (MB) solution, the absorption of MB at 664nm did not decrease in the hydrogen-producing liposomes containing vitamin C ethyl ether, even after irradiation with white light, indicating that after irradiation, 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 ascorbic acid modified by ethyl etherification, and its 3-hydroxyl group is replaced by ethyl. This modification reduces the electron-donating ability of the hydroxyl group in the molecule, and the electron-donating ability is weakened.

[0055] Fig.17 The staining of human dermal papilla cells under different conditions was tested under a microscope. The cells in the logarithmic growth phase were digested and counted, and the number of cells was 1.5×10 5The cells were inoculated with 100 μg / mL of β-galactosidase staining solution at room temperature for 15 min. The cells were washed with PBS buffer for 3 times and incubated at 37 °C overnight. The cells were observed and photographed under an inverted microscope. The results showed that dihydrotestosterone induced senescence of dermal papilla cells, while hydrogen-producing liposomes HPS (500 μg / mL) encapsulated with Au nanoparticles could inhibit β-galactosidase activity and delay cell senescence under light conditions.

[0056] Fig.18 Fluorescence microscopy and flow cytometry were used to test the fluorescence of human dermal papilla cells after DCFH-DA staining under different conditions and the DCF intensity of ROS produced by human dermal papilla cells induced by H2O2 (100 μM). 5 / mL were inoculated in a 6-well plate. The experiment was 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 1:1000. After incubation for 4 h, the culture medium was removed, 1 mL of the diluted DCFH-DA solution was added to each well, and incubated in the incubator for 20 min. Then the cells were removed and rinsed three times with serum-free DMEM medium. Observed and photographed under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). The cells were digested and collected using trypsin, and the cell suspension was counted and 5×10 per well. 4 The results showed that HPS significantly reduced the intracellular ROS induced by inflammation in human hair papilla dermis cells after adding H2O2 (100 μM) and producing hydrogen under light.

[0057] Fig.19 The levels of IL-6 and IL-1α induced by dihydrotestosterone (0.2 μg / mL) in human dermal papilla cells were tested after different treatment conditions. 5The cells were inoculated at a density of 100 cells / well in a 6-well plate. The experiment was divided into a blank group, a model group (0.2 μg / mL dihydrotestosterone), an HPS group (0.2 μg / mL dihydrotestosterone + 500 μg / mL HPS), and an HPS + L group (0.2 μg / mL dihydrotestosterone + 500 μg / mL HPS + light). The cells were incubated with drugs for 24 h, and the cell supernatant was collected. The secretion of inflammatory factors in the hair papilla cells was detected according to the operating instructions of the ELISA kit.

[0058] The results showed that compared with the blank control group, the addition of dihydrotestosterone (0.2 μg / mL) significantly increased the levels of inflammatory factors IL-6 and IL-1α in hair papilla cells. Under light conditions, HPS can significantly inhibit the expression of inflammatory factors.

[0059] In summary, the present invention provides a hydrogen production system (HPS, Hydrogen Producing System) based on Au and Pt nanoparticles. In the present invention, Au and Pt nanoparticles with small size and high dispersibility were successfully prepared based on the sodium borohydride reduction method. And 2-3 μm liposomes were prepared by thin film hydration method, which were used to encapsulate Au, Pt nanoparticles and antioxidants, and successfully constructed a tightly combined hydrogen production nanoreactor, which can produce hydrogen under natural white light irradiation. Liposomes provide a unique reactor design, which can uniquely confine the reaction molecules to the nanoscale by keeping the hydrophilic reactants (antioxidants and Au / Pt NPs) in their water core and the amphiphilic reactants (bioactive compounds) in the lipid bilayer. The liposome assembly can establish an optimal reaction environment for the reaction molecules under study.

[0060] By adjusting the ratio of liposome raw materials and encapsulating antioxidants and Au / Pt NPs in them through ice bath hydration, a stable hydrogen production system based on Au and Pt nanoparticles was synthesized, which can effectively produce hydrogen after natural light irradiation and cause methylene blue to fade quickly. In addition, the developed light-driven system effectively reduces oxidative stress, revealing its great potential to reduce tissue inflammation.

[0061] Comparative Example 1: Lip with AA 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, and 18 mg of polyoxyethylene polymer (average mass 2000) were dissolved in 60 mL of ethanol and placed in a rotary evaporator at 45°C to remove the organic solvent, and the obtained film was dissolved in 100 ml of deionized water, and 6 g of L-ascorbic acid in deionized water was added to obtain liposomes containing ascorbic acid (AA).

[0062] Comparative Example 2: Au NPs (NPs) HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k DaMW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C for use.

[0063] Comparative Example 3: Liposome (Lip) 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, and 18 mg of polyoxyethylene polymer (average mass 2000) were dissolved in 60 mL of ethanol and placed in a rotary evaporator at 45° C. to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0064] Comparative Example 4: Photosensitizer-free liposomes containing AA and Au NPs (Lip-AN) HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k DaMW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C.

[0065] 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, and 18 mg of polyoxyethylene polymer (average mass 2000) were dissolved in 60 mL of ethanol and placed in a rotary evaporator at 45° C. to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0066] 10 mL of the Au nanoparticle aqueous solution obtained in the above step was mixed with 10 mL of liposomes, and 1.2 g L-ascorbic acid was added and ultrasonically hydrated in an ice bath for 30 min to prepare photosensitizer-free liposomes encapsulating AA and Au NPs.

[0067] Comparative Example 5: Lutein Liposome (Lu-Lip) 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyethylene oxide polymer (average mass 2000) and 2 mg of lutein were dissolved in 60 mL of ethanol and placed in a rotary evaporator at 45°C to remove the organic solvent. The obtained film was dissolved in 100 mL of deionized water to obtain lutein liposomes.

[0068] Comparative Example 6: Lutein liposomes containing Au NPs (Lu-Lip-N) HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k DaMW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C.

[0069] 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average mass 2000) and 2 mg of lutein were dissolved in 60 mL of ethanol and placed in a rotary evaporator at 45°C to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0070] 10 mL of the Au nanoparticle aqueous solution obtained in the above step was mixed with 10 mL of liposomes, and ultrasonically hydrated in an ice bath for 30 min to form lutein liposomes encapsulating Au NPs.

[0071] Comparative Example 7: Lutein liposomes containing AA (Lu-Lip-A) 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average mass 2000) and 2 mg of lutein were dissolved in 60 mL of ethanol and placed in a rotary evaporator at 45°C to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0072] 0.6 g of ascorbic acid was added to 10 mL of the liposomes obtained in the above step and ultrasonically hydrated in an ice bath for 30 min to obtain AA-encapsulated lutein liposomes.

[0073] Comparative Example 8: Step 1, preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k Da MW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C.

[0074] Step 2, preparation of riboflavin-containing liposomes: 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average mass 2000) and 2 mg of riboflavin were dissolved in 60 mL of ethanol, and placed in a rotary evaporator at 45°C to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0075] Step 3, preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of liposomes obtained in step 2, 1.2 g of L-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.

[0076] Comparative Example 9: Step 1, preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k Da MW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C for use.

[0077] Step 2, preparation of resveratrol-containing liposomes: 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average mass 2000) and 2 mg of resveratrol were dissolved in 60 mL of ethanol, and placed in a rotary evaporator at 45° C. to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0078] Step 3, preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of 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.

[0079] Comparative Example 10: Step 1, preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k Da MW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C for use.

[0080] Step 2, preparation of lutein-containing liposomes: 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average mass 2000) and 2 mg of lutein were dissolved in 60 mL of ethanol, and placed in a rotary evaporator at 45°C to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0081] Step 3, preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of liposomes obtained in step 2, 1.2 g of sodium isoascorbate 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.

[0082] Comparative Example 11: Step 1, preparation of Au NPs: HAuCl4·4H2O was added to a round-bottom flask in an ice-water bath, and deionized water was added to the flask and stirred rapidly to prepare a 0.1 mM chloroauric acid aqueous solution; 0.5 mL of the chloroauric acid aqueous solution was added, 10 mL of water was added, 50 mg of Tween 80 was added as a stabilizer, and stirred for 20 minutes, and 2.5 mL of 2.4 mg / mL NaBH4 reducing agent was gradually added dropwise. After 1 hour, the liquid was placed in a 14k Da MW dialysis bag and dialyzed with deionized water for 24 hours to prepare an Au nanoparticle aqueous solution, which was stored in a refrigerator at 4°C.

[0083] Step 2, preparation of lutein-containing liposomes: 93 mg of dipalmitoylphosphatidylcholine, 22.5 mg of cholesterol, 3 mg of Tween, 18 mg of polyoxyethylene polymer (average mass 2000) and 2 mg of lutein were dissolved in 60 mL of ethanol, and placed in a rotary evaporator at 45°C to remove the organic solvent, and the obtained film was dissolved in 100 mL of deionized water to obtain liposomes.

[0084] Step 3, preparation of hydrogen-producing liposomes encapsulating Au nanoparticles: 10 mL of liposomes obtained in step 2, 1.2 g of vitamin C ethyl ether 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.

[0085] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing hydrogen-producing liposomes, characterized in that: include, Preparation of nanoparticle solution: adding gold salt, platinum salt or silver salt to a solvent, adding a stabilizer and a reducing agent to react, and obtaining a nanoparticle solution after dialysis; Preparing liposomes containing bioactive compounds; the bioactive compounds include one or more of lutein, vitamin E, coumarin, carotenoids, resveratrol, lycopene, riboflavin, curcumin, and rheum; Preparation of hydrogen-producing liposomes: mixing the liposomes containing the bioactive compound, the antioxidant and the nanoparticle solution and then ultrasonically treating them to obtain hydrogen-producing liposomes; the antioxidant comprises 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 and sodium sulfite.

2. The method for preparing hydrogen-producing liposomes according to claim 1, characterized in that: 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.

3. The method for preparing hydrogen-producing liposomes according to claim 1 or 2, characterized in that: The solvent includes water, the reaction temperature is 0-8°C, and the reaction time is 0.5-2h.

4. The method for preparing hydrogen-producing liposomes according to claim 1 or 2, characterized in that: The stabilizer includes Tween 80, and the concentration of Tween 80 is 0.01-1wt%; the reducing agent includes sodium borohydride, and 20% of 1-10 mg / mL sodium borohydride is added; the dialysis time is 6-24 hours.

5. The method for preparing hydrogen-producing liposomes according to claim 1 or 2, characterized in that: The liposomes include several of dipalmitoylphosphatidylcholine, cholesterol, 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine, Tween, sodium deoxycholate, polyethylene glycol 2000, octadecylamine, sodium taurocholate, polyoxyethylene polymer, dioleoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine polyethylene glycol 2000.

6. The method for preparing hydrogen-producing liposomes according to claim 5, characterized in that: Dipalmitoylphosphatidylcholine, 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 0.02-2wt%; wherein the concentration of the bioactive compound is 0.0001-0.005%; wherein the mass ratio of dipalmitoylphosphatidylcholine, cholesterol, Tween and polyoxyethylene polymer is 15-25:4-5:0.3-5:3-8.

7. The method for preparing hydrogen-producing liposomes according to claim 6, characterized in that: The removing of the solvent comprises removing the solvent by rotary evaporation at a temperature of 45°C.

8. The method for preparing hydrogen-producing liposomes according to claim 1 or 2, characterized in that: In step 3, the mass ratio of the liposome containing the bioactive compound to the nanoparticle solution is 1:0.5-2; and the concentration of the antioxidant is 0.5-20wt%.

9. The hydrogen-producing liposome prepared by the method for preparing the hydrogen-producing liposome according to claim 1.

10. Use of the hydrogen-producing liposomes prepared by the method for preparing the hydrogen-producing liposomes according to claim 1 in hydrogen-producing, anti-inflammatory, anti-oxidative and / or anti-hair loss products.

Images