A method for preparing human serum albumin-selenium nanoparticles loaded with chlorogenic acid

Abstract

The present invention discloses a method for preparing human serum albumin-selenium nanoparticles coated with chlorogenic acid, comprising the following steps: S1, using chlorogenic acid and human serum albumin as raw materials, preparing human serum albumin coated with chlorogenic acid by coupling reaction; S2, using the human serum albumin coated with chlorogenic acid prepared in step S1 as a dispersant to prepare human serum albumin-selenium nanoparticles coated with chlorogenic acid. The present invention discloses a method for preparing human serum albumin-selenium nanoparticles coated with chlorogenic acid, which uses human serum albumin and a small drug molecule chlorogenic acid for coupling, and then uses it as a dispersant to stabilize nano-selenium, which can not only prevent nano-selenium from aggregating and improve its stability, but also can efficiently deliver the small drug molecule chlorogenic acid, improve its water solubility and bioavailability; more importantly, it can also enable nano-selenium and the small drug molecule chlorogenic acid to synergistically exert their therapeutic functions.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a method for preparing human serum albumin-selenium nanoparticles coated with chlorogenic acid. Background Art

[0002] Selenium is an essential trace element for the human body and the active center of selenoprotein. It plays an important role in the energy metabolism and gene expression of organisms and is of great significance to human health. In nature, selenium mainly exists in three forms, namely organic selenium, inorganic selenium and nano-selenium. Among them, nano-selenium in the 0-valent state has the characteristics of lower toxicity than organic selenium and inorganic selenium, and has anti-inflammatory, antioxidant and anti-cancer effects, and has become a very potential material in the field of nanomedicine. Existing research mainly uses chemical synthesis to synthesize nano-selenium, and a redox reaction occurs with a reducing agent such as sodium selenite and ascorbic acid to obtain nano-selenium in the 0-valent state. However, nano-selenium in the 0-valent state is insoluble in water and is very easy to agglomerate, and has the disadvantage of being unstable, resulting in its particle size increasing to micrometer level, causing its bioavailability to decrease. With the gradual development of nano-drug technology, researchers have found that the selection of suitable dispersants, such as polysaccharides, proteins or natural organic components, can stabilize nano-selenium particles and improve their water solubility, thereby playing a more effective role.

[0003] Chlorogenic acid is a natural extract of honeysuckle. It is a phenolic acid generated by caffeic acid and quinic acid. It is a phenylpropanoid compound produced by plants through the shikimic acid pathway during aerobic respiration. Chlorogenic acid is an effective phenolic antioxidant with antioxidant effects, protecting tissues from oxidative damage. In addition, chlorogenic acid is also an important biologically active substance that can effectively play antibacterial, antiviral, antioxidant and anti-inflammatory effects. However, chlorogenic acid has poor water solubility and is easily metabolized in the body, resulting in low bioavailability and poor effect.

[0004] However, there are currently no reports on using protein-drug small molecule conjugates as dispersants to stabilize nano-selenium and improve the delivery efficiency of drug small molecules. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for preparing human serum albumin-selenium nanoparticles coated with chlorogenic acid. After coupling human serum albumin with the small molecule drug chlorogenic acid, the conjugate is used as a dispersant to stabilize nano-selenium, which can not only prevent nano-selenium from aggregating and improve its stability, but also efficiently deliver the small molecule drug chlorogenic acid and improve its water solubility and bioavailability. More importantly, nano-selenium and the small molecule drug chlorogenic acid can also synergistically exert their therapeutic functions.

[0006] To achieve the above object, the present invention provides a method for preparing human serum albumin-selenium nanoparticles coated with chlorogenic acid, comprising the following steps:

[0007] S1. Using chlorogenic acid and human serum albumin as raw materials, preparing human serum albumin coated with chlorogenic acid through coupling reaction;

[0008] S2. Using the chlorogenic acid-coated human serum albumin prepared in step S1 as a dispersant, prepare chlorogenic acid-coated human serum albumin-selenium nanoparticles.

[0009] Preferably, the coupling reaction in step S1 comprises the following steps:

[0010] S1-1, dissolve chlorogenic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in N,N-dimethylformamide, and react with stirring at room temperature in the dark for 24 hours;

[0011] S1-2, dissolving human serum albumin in phosphate buffer, stirring thoroughly at room temperature to dissolve, slowly and evenly dripping the solution obtained in step S1-1 while stirring, and reacting with stirring at room temperature in the dark for 12 hours;

[0012] After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed for 3 days using phosphate buffer as the dialysate. The dialysate is replaced every 12 hours to obtain purified human serum albumin coated with chlorogenic acid.

[0013] Preferably, in step S1-1, the mass volume ratio of chlorogenic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide: N,N-dimethylformamide is 18 mg: 48 mg: 44 mg: 3 mL.

[0014] Preferably, in step S1-2, the mass volume ratio of human serum albumin:phosphate buffer is 25 mg:7 mL.

[0015] Preferably, in step S2, the method for preparing chlorogenic acid-loaded human serum albumin-selenium nanoparticles comprises the following steps:

[0016] S2-1, adding 3 mg / mL sodium selenite solution to the chlorogenic acid-loaded human serum albumin solution, and stirring thoroughly at room temperature to mix;

[0017] S2-2, slowly and uniformly dripping 120 mg / mL L-ascorbic acid solution into the mixed solution of step S2-1, stirring at room temperature until the solution turns orange-red;

[0018] S2-3. Collect the reaction solution of step S2-2, retain the precipitate after centrifugation, wash it twice with a borate buffer solution with pH = 8.2 and once with ultrapure water, filter it through a 0.22 μm water filter membrane, centrifuge the filtrate again, and resuspend the precipitate in 1 mL of ultrapure water to obtain purified human serum albumin-selenium nanoparticles coated with chlorogenic acid.

[0019] Preferably, in step S2-1, the volume ratio of the sodium selenite solution to the chlorogenic acid-loaded human serum albumin solution is 2:3.

[0020] Preferably, in step S2-2, the volume ratio of L-ascorbic acid solution: mixed solution is 1:25.

[0021] Preferably, in step S2-3, the centrifugation conditions are 15000 rpm and 5 min.

[0022] The present invention provides a method for preparing chlorogenic acid-loaded human serum albumin-selenium nanoparticles, which has the following beneficial effects:

[0023] (1) For the first time, a conjugate of human serum albumin and the drug small molecule chlorogenic acid was used as a dispersant to stabilize nano-selenium;

[0024] (2) Human serum albumin-selenium nanoparticles loaded with chlorogenic acid achieve efficient delivery of the small molecule chlorogenic acid, improving its water solubility and bioavailability, thereby better exerting the therapeutic effect;

[0025] (3) Human serum albumin-selenium nanoparticles loaded with chlorogenic acid cleverly combine the drug small molecule chlorogenic acid with nano-selenium, making it have antioxidant and anti-inflammatory capabilities in both model cells and model mice, thereby synergistically exerting therapeutic functions;

[0026] (4) The drug small molecule chlorogenic acid and nano-selenium exert different enzyme-like activities respectively, and the prepared human serum albumin-selenium nanoparticles loaded with chlorogenic acid can organically combine the two to synergistically exert the optimal antioxidant effect;

[0027] (5) Chlorogenic acid-loaded human serum albumin-selenium nanoparticles can exert enzyme-like activity to remove overproduced reactive oxygen species, thereby improving the oxidative stress environment;

[0028] (6) Human serum albumin-selenium nanoparticles loaded with chlorogenic acid can reduce the expression of pro-inflammatory cytokines, thereby exerting an anti-inflammatory effect;

[0029] (7) The prepared nanoparticles have uniform particle size, high stability and good biosafety;

[0030] (8) The chemical synthesis technology of nano-selenium is mature; the synthesis method is simple and easy to operate; the synthetic raw materials are easy to obtain and inexpensive.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the preparation process of human serum albumin-selenium nanoparticles loaded with chlorogenic acid;

[0033] Figure 2 Zeta surface potential diagram (A) and ultraviolet absorption spectrum diagram (B) of chlorogenic acid, human serum albumin and human serum albumin coated with chlorogenic acid, and matrix-assisted laser dissociation ionization-time of flight mass spectrometry diagram of human serum albumin (C) and human serum albumin coated with chlorogenic acid (D);

[0034] Figure 3 Transmission electron microscopy image (A) of human serum albumin-selenium nanoparticles loaded with chlorogenic acid and element distribution map of nanoparticles, where B is a dark field image of nanoparticles, CE images are the distribution of Se, C, and N elements, and F is a Merge image of dark field and Se;

[0035] Figure 4 The ultraviolet absorption spectra of nano-selenium, human serum albumin coated with chlorogenic acid, and human serum albumin-selenium nanoparticles coated with chlorogenic acid (A) and the X-ray photoelectron spectrum of human serum albumin-selenium nanoparticles coated with chlorogenic acid (B);

[0036] Figure 5 The particle size results of human serum albumin-selenium nanoparticles coated with chlorogenic acid (A), the Zeta surface potential diagram of selenium, human serum albumin coated with chlorogenic acid, and human serum albumin-selenium nanoparticles coated with chlorogenic acid (B), and the particle size changes of human serum albumin-selenium nanoparticles coated with chlorogenic acid in three different solvents (C);

[0037] Figure 6 A bar graph of nanoparticle cytotoxicity analysis showing the calculation of cell survival rates in different groups after RAW264.7 cells were treated with chlorogenic acid-loaded human serum albumin-selenium nanoparticles at different selenium and chlorogenic acid concentrations;

[0038] Figure 7 To detect the synergistic antioxidant effect test results of human serum albumin-selenium nanoparticles coated with chlorogenic acid, wherein A is the SOD-like enzyme activity of different groups, B is the SOD-like enzyme activity of human serum albumin-selenium nanoparticles coated with chlorogenic acid at different concentrations, C is the GPX-like enzyme activity of different groups, and D is the GPX-like enzyme activity of human serum albumin-selenium nanoparticles coated with chlorogenic acid at different concentrations;

[0039] Figure 8RAW264.7 cells in different treatment groups were stained with ROS FITC + Statistical graph of cells;

[0040] Fig. 9 are the expression levels of proinflammatory cytokines at the gene level in RAW264.7 cells in different treatment groups, where A is the expression level of interleukin-1β (IL-1β), B is the expression level of interleukin-6 (IL-6), and C is the expression level of tumor necrosis factor-ɑ (TNF-ɑ);

[0041] Fig.10 are the antioxidant levels in the lungs of mice in different treatment groups, where A is SOD enzyme activity and B is GPX enzyme activity;

[0042] Fig.11 Are the expression levels of proinflammatory cytokines in the serum of mice in different treatment groups, where A is the expression level of IL-1β, B is the expression level of IL-6, and C is the expression level of TNF-ɑ. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention clearer, the embodiments of the present invention are further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions.

[0044] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or server that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units that are not explicitly listed or inherent to these processes, methods, products or devices.

[0045] Like reference numerals and letters denote similar items in the following drawings, and thus, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0046] Embodiment 1

[0047] A method for preparing human serum albumin-selenium nanoparticles loaded with chlorogenic acid, such as Figure 1 As shown, the specific steps include:

[0048] (1) Weigh 18 mg of chlorogenic acid (CGA), 48 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 44 mg of N-hydroxysuccinimide (NHS), dissolve in 3 mL of N,N-dimethylformamide, and react with stirring at room temperature in the dark for 24 h.

[0049] (2) Weigh 25 mg of human serum albumin (HSA) and dissolve it in 7 mL of phosphate buffer. Stir thoroughly to dissolve it at room temperature. Slowly and evenly add the solution obtained in the above (1) while stirring. Stir and react for 12 h at room temperature in the dark.

[0050] (3) After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed for 3 days using phosphate buffer as the dialysate. The dialysate is replaced every 12 hours to obtain purified chlorogenic acid-loaded human serum albumin (CGA-HSA), which is then stored at 4°C for later use.

[0051] (4) Take 3 mL of CGA-HSA and stir thoroughly at room temperature.

[0052] (5) Prepare a 3 mg / mL sodium selenite solution, pipette 2 mL into the above solution, and stir thoroughly at room temperature to mix.

[0053] (6) Prepare a 120 mg / mL L-ascorbic acid solution, pipette 200 μL of it and slowly and uniformly drip it into the above mixed solution. Stir and react at room temperature for 30 min. When the solution gradually turns orange-red, the reaction is complete.

[0054] (7) The reaction solution was collected and centrifuged at 15,000 rpm for 5 min for purification. After centrifugation, the precipitate was retained and washed twice with a borate buffer solution at pH 8.2 and once with ultrapure water. The precipitate was then filtered through a 0.22 μm water filter membrane. The filtrate was centrifuged again and the precipitate was resuspended in 1 mL of ultrapure water to obtain purified chlorogenic acid-loaded human serum albumin-selenium nanoparticles (CGA-HSA-Se), which were then stored at 4°C for later use.

[0055] Experimental Testing

[0056] (1) Figure 2 As shown, Figure 2 Figure A in the middle is the Zeta surface potential diagram. From the figure, it can be seen that the potential of CGA is about +10mV, the potential of HSA is about -16mV, and the potential of CGA-HSA is about -24mV. The potential has changed, proving the successful synthesis of CGA-HSA.

[0057] Figure 2Figure B is the UV absorption spectrum. It can be seen that HSA has a maximum absorption peak at 280nm, CGA has a maximum absorption peak at 335nm, and CGA-HSA has the characteristic absorption peaks of both CGA and HSA, and a red shift occurs at 335nm, proving that an amidation reaction occurred between CGA and HSA to form a new amide bond, which also proves the successful synthesis of CGA-HSA.

[0058] Figure 2 Figures C and D are matrix-assisted laser dissociation ionization-time of flight mass spectrometry. Figure C is the relative molecular mass of HSA, which is 67175.201, and Figure D is the relative molecular mass of CGA-HSA, which is 76521.927. The relative molecular mass of CGA is known to be 354.309. Substituting it into the following formula, the coupling rate Mr of CGA coupled to one molecule of HSA can be calculated to be 26.

[0059]

[0060] (2) Figure 3 As shown, Figure 3 In Figure A, the morphology of CGA-HSA-Se was analyzed by transmission electron microscopy. It can be seen that the nanoparticles have uniform particle size, good dispersion, and a layer of film on the surface, indicating that CGA-HSA successfully attached to the surface of nano-selenium to form a film.

[0061] Transmission electron microscopy and energy dispersive X-ray spectroscopy were then used together, e.g. Figure 3 As shown in Figure BF, the surface scan element distribution is the element distribution map of the nanoparticles, Figure B is the dark field map of the particles, CE maps are the distribution of Se, C, and N elements respectively, and Figure F is the Merge map of dark field and Se. It can be seen that Se is distributed in the center of the particle to form a "core", while C and N elements are distributed on the surface of Se to form a "shell", proving that CGA-HSA is successfully attached to the surface of nano-selenium to form a typical "core-shell" structure.

[0062] (3) Figure 4 As shown, Figure 4 Figure A in the middle is an ultraviolet absorption spectrum. It can be seen that nano-selenium (Se) has a maximum absorption peak at 262nm, CGA-HSA has characteristic absorption peaks at 280nm and 313nm respectively, and CGA-HSA-Se has overlapping peaks of Se and CGA-HSA characteristic absorption peaks, and a red shift phenomenon occurs, proving the successful synthesis of CGA-HSA-Se.

[0063] Figure 4Figure B is the X-ray photoelectron spectrum (XPS) of CGA-HSA-Se. The nanoparticles were freeze-dried to obtain a powder sample. Al Ka ​​was used as the X-ray source, and the binding energy was calibrated using the contaminated carbon C1s as the correction value. The vacuum condition was 5×10 -8 Pa, and then the elemental composition of the sample was determined on the machine. The results showed that the characteristic peaks corresponding to Se3d, C1s, N1s and O1s all existed, indicating that CGA-HSA-Se was mainly composed of Se, C, N, and O elements, further confirming the successful synthesis of CGA-HSA-Se.

[0064] (4) Figure 5 As shown, Figure 5 Figure A in the middle is the particle size result diagram of CGA-HSA-Se. It can be seen from the figure that the particle size of the nanoparticles is about 35nm and the particle size is uniform.

[0065] Figure 5 Figure B in the middle is the Zeta surface potential. It can be seen from the figure that the potential of CGA-HSA is about -24 mV, the potential of Se is about -30 mV, and the potential of CGA-HSA-Se is about -24 mV, which is consistent with the potential of CGA-HSA, proving that CGA-HSA successfully attaches to the surface of Se to form a film, proving the successful synthesis of CGA-HSA-Se.

[0066] Figure 5 Figure C in the middle shows the particle size changes of CGA-HSA-Se in three different solvents (ultrapure water ddH2O, phosphate buffer PBS, and culture medium DMEM) within one month. It can be seen that the particle size of the nanoparticles hardly changes within one month, and has excellent stability.

[0067] (5) Figure 6 In order to test the toxicity of CGA-HSA-Se to mouse mononuclear macrophage leukemia cells (RAW264.7 cells) using the MTT method, the biosafety of the nanoparticles was evaluated. Cells were treated with CGA-HSA-Se at different concentrations (Se concentration was 1, 10, 100, 200, 400, 600 ng / mL; CGA concentration was 1.22, 12.2, 122, 244, 488, 732 ng / mL), and the MTT cell proliferation and cytotoxicity detection kit was followed. The cell survival rate of each group relative to the blank control group (Se concentration and CGA concentration were 0) was analyzed and calculated by the user.

[0068] Cell survival rate (%) = absorbance value of experimental cell group (A2) / absorbance value of blank cell group (A1) × 100%.

[0069] like Figure 6As shown, the cell survival rates of different groups of cells were calculated according to the different concentrations of CGA-HSA-Se treated cells to make the cytotoxicity analysis bar graph of the nanoparticles. The results show that the concentration of CGA-HSA-Se is basically linearly related to its cytotoxicity to RAW264.7 cells. Although the higher the concentration, the greater the toxicity to the cells, when the Se concentration reaches 600ng / mL (higher concentration), the survival rate of RAW264.7 cells remains above 75%, which shows that the nanoparticles of the present invention have good biosafety.

[0070] (6) Existing studies have shown that the moderate production of reactive oxygen species (ROS) in cells is part of the normal physiological process. ROS has a strong oxidative capacity and can resist the invasion of pathogenic microorganisms or viruses during the immune process. It is beneficial to the organism. However, when ROS are produced excessively or insufficiently cleared, it will cause oxidative stress, causing damage to normal cells, DNA, proteins, etc., leading to inflammation and the occurrence of many diseases. Therefore, clearing excessive ROS and exerting an antioxidant effect is one of the important ways to fight inflammation and anti-aging. Common antioxidant enzymes in organisms include superoxide dismutase (SOD) and glutathione peroxidase (GPX), which can clear excessive ROS and exert an antioxidant effect, thereby achieving the effect of treating inflammation and ROS-related diseases.

[0071] In order to detect the synergistic antioxidant effect of CGA-HSA-Se, different experimental groups were designed, including control group, chlorogenic acid treatment group (CGA), human serum albumin-selenium nanoparticles treatment group (HSA-Se), and human serum albumin-selenium nanoparticles loaded with chlorogenic acid (CGA-HSA-Se). The superoxide dismutase (SOD) activity detection kit and glutathione peroxidase (GSH-Px / GPX) activity detection kit were used to detect the SOD-like and GPX-like enzyme activities of CGA-HSA-Se in different groups and different concentrations (Se concentration was 5, 10, 20, 50, 100, 200 μg / mL) in vitro.

[0072] Xanthine and xanthine oxidase can generate O 2- , O 2- It can reduce nitroblue tetrazolium to generate blue formazan, which has a characteristic absorption peak at 560nm; SOD enzyme can remove O 2-, inhibiting the formation of formazan, thereby reducing the ultraviolet absorbance at 560nm, and the decrease in absorbance at 560nm can reflect the activity of the SOD enzyme. According to the steps in the kit instructions, after the reaction is completed, the absorbance of different groups of reaction solutions at 560nm is detected by a UV spectrophotometer. GPX enzyme can catalyze reduced glutathione (GSH) to generate oxidized glutathione (GSSG), reducing toxic hydrogen peroxide to non-toxic hydroxyl compounds (DTNB), which have a characteristic absorption peak at 412nm, and the decrease in absorbance at 412nm can reflect the activity of the GPX enzyme. According to the steps in the kit instructions, after the reaction is completed, the absorbance of different groups of solutions at 412nm is detected by a UV spectrophotometer. In addition, CGA-HSA-Se was gradiently diluted into solutions of different concentrations (Se concentration was 5, 10, 20, 50, 100, and 200 μg / mL) and operated according to the instructions. After the reaction was completed, the absorbance of the solution at 560 nm (SOD enzyme) and 412 nm (GPX enzyme) was detected by an ELISA instrument, and the O treated with different concentrations of CGA-HSA-Se was analyzed and calculated. 2- and GSH clearance rate, that is, SOD-like and GPX enzyme activities.

[0073] like Figure 7 As shown, Figure 7 Figure A in the middle shows the SOD-like enzyme activity of different groups. It can be observed that compared with the control group, the absorbance of the CGA, HSA-Se and CGA-HSA-Se groups decreased, and the absorbance value of CGA-HSA-Se was the lowest, indicating that compared with the CGA group and the HSA-Se group, the O 2- The clearing effect is the most obvious and the SOD-like enzyme activity is the strongest.

[0074] Figure 7 Figure B shows the SOD-like enzyme activity of CGA-HSA-Se at different concentrations. It can be seen that with the increase of CGA-HSA-Se concentration, 2- The clearance rate gradually increased and the activity of SOD-like enzymes gradually became stronger, showing significant concentration dependence.

[0075] Figure 7 Figure C in the middle shows the GPX-like enzyme activity of different groups. It can be observed that compared with the control group, the absorbance of the CGA, HSA-Se and CGA-HSA-Se groups decreased, and the absorbance value of CGA-HSA-Se was the lowest, indicating that compared with the CGA group and the HSA-Se group, the CGA-HSA-Se group had the most obvious GSH scavenging effect and the strongest GPX-like enzyme activity.

[0076] Figure 7Figure D in the middle shows the GPX-like enzyme activity of CGA-HSA-Se at different concentrations. It can be seen that as the concentration of CGA-HSA-Se increases, the GSH clearance rate gradually increases and the GPX-like enzyme activity gradually becomes stronger, showing significant concentration dependence.

[0077] exist Figure 7 In A, the absorbance of the CGA group decreased significantly, while that of the HSA-Se group hardly changed; Figure 7 In Figure C, the absorbance of the HSA-Se group decreased significantly, while the absorbance of the CGA group hardly changed. The absorbance of CGA-HSA-Se was the lowest in both tests. This shows that CGA has SOD-like enzyme activity but almost no GPX-like enzyme activity, while HSA-Se has GPX-like enzyme activity but almost no SOD-like enzyme activity. CGA-HSA-Se cleverly combines CGA with HSA-Se, making it have both SOD-like enzyme activity and GPX-like enzyme activity, thereby synergistically exerting a more efficient antioxidant effect.

[0078] (7) In order to verify the antioxidant effect of CGA-HSA-Se in cells, lipopolysaccharide (LPS) was used to stimulate ROS overexpression in RAW264.7 cells, leading to oxidative stress. The cells were then treated with HSA-Se, CGA, and CGA-HSA-Se, respectively. The cells were stained with the ROS green fluorescent probe DCFH-DA and detected by flow cytometry. ROSFITC + Statistical analysis of cells.

[0079] like Figure 8 As shown in the figure, the experimental group was divided into 5 groups, namely, the control group (control group: no treatment), LPS group, HSA-Se group, CGA group and CGA-HSA-Se group. Compared with the control group, the intracellular ROS level in the LPS group increased by 4 times, and ROS FITC + However, after treatment with HSA-Se, CGA, and CGA-HSA-Se, ROS FITC + The percentages of ROS FITC in the CGA-HSA-Se group were reduced to 46.9%, 41.2% and 33.4%, respectively. + The cell ratio was the lowest, which confirmed that CGA-HSA-Se had a stronger ROS scavenging ability than HSA-Se and CGA, and could play a more effective antioxidant role in cells.

[0080] (8) Overactivated macrophages secrete a large amount of proinflammatory cytokines, which further accelerate the development of intracellular inflammatory responses. In order to verify the anti-inflammatory effect of CGA-HSA-Se in cells, RAW264.7 cells were also activated with LPS, and then treated with HSA-Se, CGA, and CGA-HSA-Se. The expression levels of proinflammatory cytokines at the gene level, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-ɑ (TNF-ɑ), were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) technology.

[0081] like Fig. 9 As shown in the figure, the experimental group was also divided into 5 groups, namely, the Control group (control group: no treatment), LPS group, HSA-Se group, CGA group and CGA-HSA-Se group. After LPS treatment, the expression level of Il-1β in RAW264.7 cells was significantly higher than that in the Control group. Compared with the LPS group, after treatment with HSA-Se, CGA and CGA-HSA-Se, the expression level of Il-1β decreased to varying degrees, and the expression level of the CGA-HSA-Se group decreased the least ( Fig. 9 A), and Il-6( Fig. 9 Chinese B), Tnf-ɑ( Fig. 9 The expression level in (C) also showed the same trend.

[0082] The above results show that HSA-Se, CGA, and CGA-HSA-Se can all inhibit the expression of proinflammatory cytokines, and CGA-HSA-Se has a higher inhibition level compared with HSA-Se and CGA, and can exert anti-inflammatory effects more efficiently in cells.

[0083] (9) During the development of inflammation, a large amount of ROS will be generated. The antioxidant capacity of antioxidant enzymes in the inflammatory site is insufficient, and ROS clearance is not timely, which will trigger oxidative stress and aggravate the inflammatory response. In order to explore the antioxidant effect of CGA-HSA-Se in mice, an LPS-induced acute lung injury model was established in mice, and HSA-Se, CGA, and CGA-HSA-Se were used for treatment. Subsequently, the lungs of the mice were removed, and the antioxidant enzyme activity in the lungs of different groups of mice was detected using a superoxide dismutase (SOD) activity detection kit and a reduced glutathione (GSH) assay kit. The operations were performed according to the steps in the kit instructions to explore the antioxidant effect of CGA-HSA-Se in mice.

[0084] like Fig.10As shown, the experimental group was divided into 5 groups, namely, Control group (control group: no treatment), LPS group, HSA-Se group, CGA group and CGA-HSA-Se group. Fig.10 A in the middle is the activity of SOD enzyme in different groups. From the results, it can be seen that the SOD enzyme activity in the LPS group was significantly lower than that in the Control group, indicating that oxidative stress led to the downregulation of SOD enzyme activity. Compared with the LPS group, the SOD level was slightly increased after treatment with HSA-Se and CGA, but there was no significant change, while CGA-HSA-Se significantly increased the SOD level, indicating that CGA-HSA-Se has higher SOD enzyme activity than HSA-Se and CGA.

[0085] Fig.10 Figure B shows the GSH content of different groups. The level of GSH content can reflect the activity of GPX enzyme. Compared with the Control group, the GSH content was significantly reduced after LPS induction, which also indicates that oxidative stress downregulates the activity of GPX enzyme. It is worth noting that compared with the LPS group, the GSH levels of the HSA-Se and CGA groups were slightly increased, while the GSH level of the CGA-HSA-Se group was the highest, confirming that CGA-HSA-Se has higher GPX enzyme activity than HSA-Se and CGA.

[0086] In summary, the above results prove that CGA-HSA-Se can exert excellent antioxidant effects in vivo and thus reduce the level of oxidative stress in mice.

[0087] (10) To further verify the anti-inflammatory effect of CGA-HSA-Se in mice, the LPS-induced acute lung injury model in mice was established, and then they were treated with HSA-Se, CGA, and CGA-HSA-Se, respectively. The expression levels of serum proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in different groups of mice were analyzed by enzyme-linked immunosorbent assay (ELISA) technology.

[0088] like Fig.11 As shown, the experimental group was also divided into 5 groups, namely, the Control group (control group: no treatment), LPS group, HSA-Se group, CGA group and CGA-HSA-Se group. Compared with the Control group, the expression level of IL-1β in the LPS group was significantly increased. After treatment with HSA-Se, CGA and CGA-HSA-Se, the expression level of IL-1β decreased. Among them, the expression level of IL-1β in the CGA-HSA-Se group was the lowest, indicating that it has the best effect in reducing the expression level of IL-1β ( Fig.11 Middle A). And IL-6 ( Fig.11 Middle B), TNF-α( Fig.11 The expression level in (C) also showed the same trend.

[0089] The above results show that HSA-Se, CGA and CGA-HSA-Se can inhibit the expression of proinflammatory cytokines, and CGA-HSA-Se has a higher inhibition level compared with HSA-Se and CGA, and can exert a more efficient anti-inflammatory effect in mice.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution 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 they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing human serum albumin-selenium nanoparticles coated with chlorogenic acid, characterized in that: The steps include: S1. Using chlorogenic acid and human serum albumin as raw materials, preparing human serum albumin coated with chlorogenic acid through coupling reaction; S2, using the chlorogenic acid-coated human serum albumin prepared in step S1 as a dispersant to prepare chlorogenic acid-coated human serum albumin-selenium nanoparticles; The coupling reaction in step S1 comprises the following steps: S1-1, dissolve chlorogenic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in N,N-dimethylformamide, and react with stirring at room temperature in the dark for 24 hours; S1-2, dissolving human serum albumin in phosphate buffer, stirring thoroughly at room temperature to dissolve, slowly and evenly dripping the solution obtained in step S1-1 while stirring, and reacting with stirring at room temperature in the dark for 12 hours; After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed for 3 days using phosphate buffer as the dialysate. The dialysate is changed every 12 hours to obtain purified human serum albumin coated with chlorogenic acid; In step S1-1, the mass volume ratio of chlorogenic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide: N,N-dimethylformamide is 18 mg: 48 mg: 44 mg: 3 mL; In step S1-2, the mass volume ratio of human serum albumin: phosphate buffer is 25 mg: 7 mL; In step S2, the method for preparing human serum albumin-selenium nanoparticles loaded with chlorogenic acid comprises the following steps: S2-1, adding 3 mg / mL sodium selenite solution to the chlorogenic acid-loaded human serum albumin solution, and stirring thoroughly at room temperature to mix; S2-2, slowly and uniformly dripping 120 mg / mL L-ascorbic acid solution into the mixed solution of step S2-1, stirring at room temperature until the solution turns orange-red; S2-3. Collect the reaction solution of step S2-2, retain the precipitate after centrifugation, wash it twice with a borate buffer solution of pH = 8.2 and once with ultrapure water, filter it through a 0.22 μm water filter membrane, centrifuge the filtrate again, and resuspend the precipitate in 1 mL of ultrapure water to obtain purified human serum albumin-selenium nanoparticles coated with chlorogenic acid; In step S2-1, the volume ratio of sodium selenite solution to chlorogenic acid-loaded human serum albumin solution is 2:3; In step S2-2, the volume ratio of L-ascorbic acid solution: mixed solution is 1:

25.

2. The method for preparing chlorogenic acid-loaded human serum albumin-selenium nanoparticles according to claim 1, characterized in that: In step S2-3, the centrifugation conditions are 15000 rpm and 5 min.

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