CN109833329B - Hyaluronic acid-porous nano selenium compound and preparation method and application thereof - Google Patents
Hyaluronic acid-porous nano selenium compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nano materials, and discloses a hyaluronic acid-porous nano selenium compound and a preparation method and application thereof. Specifically, the porous nano-selenium aqueous solution is mixed with EDC and NHS for activation, and then the activated porous nano-selenium aqueous solution is mixed with HA for reaction to obtain the hyaluronic acid-porous nano-selenium compound aqueous solution. The nano selenium with the super-large specific surface area and the porous structure can eliminate active oxygen to inhibit inflammation and oxidation damage, has higher affinity and reactivity to the active oxygen compared with the traditional nano selenium, and enhances the electron affinity and conduction efficiency of the active oxygen and the nano selenium. And through the modification of HA, the targeting property of the porous selenium to inflammatory macrophages is improved, and the drug intake of cells can be increased, so that the intracellular drug is maintained at a higher level. Enhances the activity of the porous selenium for eliminating active oxygen in inflammatory cells and in vivo, and provides a thought for the functional modification of selenium antioxidants.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a hyaluronic acid-porous nano selenium compound and a preparation method and application thereof.
Background
Inflammation is a typical pathological feature present in many diseases, such as bacterial infections, cancer, and atherosclerosis. In sepsis in particular, systemic inflammation can trigger the risk of multiple organ failure leading to acute death. Studies have found that a large number of activated macrophages infiltrate the inflammatory microenvironment, producing an excess of activated macrophagesReactive oxygen species, causing extracellular toxicity. The active oxygen mainly comprises superoxide anion (.O)2-, hydroxyl radical (. OH) and hydrogen peroxide (H)2O2) Excessive reactive oxygen species tend to damage normal cellular nucleic acids, liposomes and proteins, causing severe damage. Therefore, the development of efficient antioxidants to inhibit inflammatory injury is an urgent need in the course of sepsis therapy and in clinical surgery. Selenium is a natural antioxidant and can protect against oxidative damage by free radicals in vivo by increasing the activity of selenium-containing reductases. In addition, with the development of nanotechnology in recent years, the unique structure and physicochemical properties of the nano material promote the research and application of nano selenium in various diseases. Research shows that the selenium nano particle has activity of eliminating hydroxyl radical, but the active oxygen eliminating efficiency is only 22% due to the low reactivity of the nano selenium. Therefore, in order to further improve the capability of eliminating active oxygen by nano-selenium, a novel targeting nano-selenium with high reactivity needs to be developed.
In addition, the low targeting to the macrophage is another important reason of low elimination efficiency of the nano-selenium active oxygen. Hyaluronic acid is a molecule specifically binding to a cell surface molecule CD44, and CD44 is a highly expressed protein molecule in macrophages, so that the hyaluronic acid molecule is an effective strategy for enhancing the targeting property of the hyaluronic acid molecule by modifying the surface of nano-selenium.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the present invention provides a method for preparing a hyaluronic acid-porous nano selenium complex.
The invention also aims to provide the hyaluronic acid-porous nano selenium compound prepared by the method.
The invention further aims to provide application of the hyaluronic acid-porous nano selenium compound in preparation of a medicine for treating septicemia.
The purpose of the invention is realized by the following scheme:
a preparation method of a hyaluronic acid-porous nano selenium compound comprises the following steps:
mixing the porous nano selenium aqueous solution with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), activating, and then mixing and reacting the activated porous nano selenium aqueous solution with Hyaluronic Acid (HA) to obtain a hyaluronic acid-porous nano selenium compound (MSe-HA) aqueous solution.
The concentration of the porous nano-selenium in the porous nano-selenium aqueous solution is 0.1-10 mg/mL; preferably 2 mg/mL.
The mol ratio of the porous nano selenium to the EDC to the NHS is 1: 0.1: 0.1-1: 20: 20; preferably 1: 1: 1-1: 2: 2.
the activation time is 0.5-10 h; preferably 3-5 h; more preferably 4 h.
The mass ratio of the porous nano selenium to the hyaluronic acid is 1: 1-1: 10; preferably 1: 3-1: 5.
the molecular weight of the hyaluronic acid is 10 KD-50 KD.
The mixing reaction condition is stirring reaction at room temperature for 6-18 h; the mixing reaction time is preferably 8-14 h; more preferably 12 h.
Preferably, the method further comprises a purification process after the mixing reaction, specifically, the solution after the mixing reaction is centrifuged, collected, precipitated, washed and then resuspended, so as to obtain MSe-HA aqueous solution; the centrifugation is carried out for 10-15 min at 8000-10000 rpm; the number of washes was 3.
The preparation method of the porous nano selenium aqueous solution comprises the following steps:
dissolving mesoporous silica powder in an organic solvent, mixing with 3-Aminopropyltriethoxysilane (APTES), performing reflux reaction to obtain an aminated mesoporous silica solution, then dropwise adding a sodium selenite aqueous solution, stirring for reaction, dropwise adding a reducing agent, continuously stirring for reaction, obtaining a reaction solution after the reaction is finished, and then adding hydrofluoric acid to etch away the mesoporous silica to obtain the porous nano selenium.
The organic solvent is at least one of absolute ethyl alcohol and toluene.
The mass-volume ratio of the mesoporous silica to the organic solvent to the APTES is 0.1 g: 5mL of: 10 mu L-20 g: 100mL of: 100 mu L of the solution; preferably 10 g: 10mL of: 50 μ L.
The reflux reaction time is 3-10 h; preferably 6 h.
The concentration of sodium selenite in the sodium selenite aqueous solution is 0.1-1.0 mol/L.
The volume ratio of the aminated mesoporous silica solution to the sodium selenite aqueous solution is 1: 2-1: 10; preferably 1: 4.
the reducing agent is at least one of citric acid, aspartic acid, glutamic acid, retinoic acid and L-ascorbic acid.
The molar ratio of the sodium selenite to the reducing agent is 1: 1-1: 5.
the time of the first stirring reaction is 15-60 min, and the time of the second stirring reaction is 1-5 h;
preferably, the time for the first stirring reaction is 30min, and the time for the second stirring reaction is 2 h.
The concentration of the hydrofluoric acid is 20-70 wt%, and preferably 40.0 wt%; the dosage of the hydrofluoric acid is 5-200 mu L for every 1mL of reaction liquid.
The mesoporous silica is prepared by a method of hydrolyzing Tetraethoxysilane (TEOS) by a Cetyl Trimethyl Ammonium Bromide (CTAB) template, and the method comprises the following specific steps:
dissolving cetyl trimethyl ammonium bromide in water, adding an organic solvent A, then adjusting the solution to be alkaline, adding tetraethoxysilane after stirring and dissolving, raising the temperature, stirring and reacting to obtain a reaction solution, and then refluxing the reaction solution in a mixed solution of an acidic solution and an organic solvent B to obtain the mesoporous silicon dioxide.
The organic solvent A is at least one of ethanol and toluene.
The mass volume ratio of the hexadecyl trimethyl ammonium bromide to the water to the organic solvent A is 50 mg: 20mL of: 5 mL-200 mg: 100mL of: 50 mL; preferably 100 mg: 68 mL: 30 mL.
The molar ratio of the ethyl orthosilicate to the hexadecyl trimethyl ammonium bromide is 1: 1-1: 10.
and adjusting the pH of the solution to be 9-10 by using ammonia water or NaOH aqueous solution.
The temperature is raised to 45-70 ℃ and then the stirring reaction is continued for 12-36 h; the reaction is preferably stirred for 24h with increasing temperature to 60 ℃.
Preferably, the stirring reaction further comprises a purification process after the stirring reaction, specifically, the solution after the stirring reaction is centrifugally washed three times; more preferably, ethanol is used for washing.
The acidic solution is HCl, and the organic solvent B is absolute ethyl alcohol; the pH value of the mixed solution of the acidic solution and the organic solvent is 1-3.
The refluxing time is 8-12 h.
The invention does not indicate that the temperature is carried out at room temperature, and the room temperature is 25-35 ℃.
The hyaluronic acid-porous nano selenium compound prepared by the method.
The hyaluronic acid-porous nano selenium compound is applied to preparation of a medicine for treating septicemia.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) compared with the traditional nano selenium, the porous selenium has higher affinity and reactivity to active oxygen and enhances the electron affinity and conduction efficiency of the active oxygen and the nano selenium, so that the porous nano selenium has high-efficiency broad-spectrum active oxygen elimination activity.
(2) The invention provides application of targeted porous nano-selenium in eliminating active oxygen to treat septicemia, effectively improves the targeting of the porous selenium to inflammatory macrophages through modification of hyaluronic acid, and can increase the drug intake of cells, thereby ensuring that intracellular drugs are maintained at a higher level. Further enhances the activity of the porous selenium for eliminating active oxygen in inflammatory cells and in vivo, and provides a thought for the functional modification of selenium antioxidants.
(3) The preparation process and the product system of the hyaluronic acid modified porous nano-selenium provided by the invention are simple, the product can be directly stored and used, and the preparation method is simple and convenient.
(4) The hyaluronic acid modified porous nano selenium provided by the invention has good stability and dispersibility in an aqueous solution, and can be stored and used in a room temperature environment. In addition, hyaluronic acid and selenium are both essential substances and elements in the human body, have good biocompatibility and have low irritation in the body. Can be degraded into non-toxic substances after reacting with active oxygen, and has good clinical application potential.
Drawings
FIG. 1 is a transmission electron micrograph and a specific surface inspection image of MSe NPs obtained in example 1, wherein A is a transmission electron micrograph of 39000 times; b is a transmission electron microscope image which is 65000 times; c is a nitrogen adsorption desorption curve chart; d is the aperture distribution diagram.
FIG. 2 is a transmission electron micrograph and a composite image of MSe-HA NPs obtained in example 1, wherein A is a transmission electron micrograph, B is an infrared spectrum, and C is an ultraviolet spectrum.
FIG. 3 is a graph showing the effect of Se NPs, MSe NPs and MSe-HA NPs obtained in example 1 on eliminating active oxygen, wherein A is a graph showing fluorescence intensity at an emission wavelength of 650 nm; b is a fluorescence intensity diagram at an emission wavelength of 510 nm; c is a fluorescence intensity map at an emission wavelength of 425 nm; d is a superoxide anion elimination efficiency graph; e is a hydroxyl radical elimination rate graph; f is a graph of hydrogen peroxide elimination rate.
FIG. 4 is a map of the MSe NPs and MSe-HA NPs obtained in example 1, in macrophage cells and mice, wherein A is the cellular uptake pattern of macrophages; b is a fluorescence quantification graph in the cell absorption of macrophages; c is a porous nano-selenium in vivo targeting map; d is a target fluorescence quantification picture in the body of the porous nano selenium.
FIG. 5 is a graph of experiments on the elimination of activated oxygen in macrophages by Se NPs, MSe NPs and MSe-HA NPs obtained in example 1, wherein A is a laser copolymerization diagram; b is a flow cytogram.
FIG. 6 is a graph of experiments on the removal of reactive oxygen species-induced sepsis in mice by Se NPs, MSe NPs and MSe-HA NPs obtained in example 1, wherein A is a graph of ear injury in mice; b is a fluorescence map of an active oxygen probe DCFH-DA; c is a fluorescence quantification graph of an active oxygen probe DCFH-DA; d is H & E section of mouse liver and lung.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
The reagents used in the examples are commercially available without specific reference.
RAW264.7 cells (chinese academy of sciences type culture collection cell bank); c57BL/6 mouse (Guangdong province animal experiment center)
Example 1 Synthesis of Nano-selenium, porous Nano-selenium and hyaluronic acid-porous Nano-selenium composite
(1) The synthesis method of porous nano selenium (MSe NPs) and nano selenium comprises the following steps: dissolving 100mg of Cetyl Trimethyl Ammonium Bromide (CTAB) in 68mL of distilled water, stirring at normal temperature, adding 30mL of ethanol, adjusting the pH value to 9-10 with ammonia water, adding 80 mu L of TEOS after stirring and dissolving, heating to 60 ℃, continuously stirring for reacting for 24h, centrifugally cleaning with absolute ethyl alcohol for three times after the reaction is finished, and refluxing in a mixed solution (pH is 1) of HCl and ethanol for 6h to obtain the mesoporous silicon dioxide. 10g of mesoporous silica powder is dissolved in 10mL of absolute ethyl alcohol, and 50 mu L of APTES is added for refluxing for 6h to obtain aminated mesoporous silica. By utilizing the property that the aminated silica can adsorb negatively charged sodium selenite due to positive charge, 5mL of mesoporous silica solution is dropwise added with 5mL of sodium selenite solution (5mg/mL) and stirred for 30min, and then 10mL of reducing agent L-ascorbic acid solution (10mg/mL) is dropwise added and stirred for 2 h. After the reaction is finished, the reaction product is centrifugally washed for three times, and 100 mu L of hydrofluoric acid is added to etch the mesoporous silicon dioxide to obtain the porous nano selenium.
(2) MSe-HA NPs synthesis method: dissolving the porous nano-selenium obtained in the step (1) in water to prepare a solution of 2mg/mL, activating the obtained solution of 10mL with EDC (30mg) and NHS (40mg) at room temperature for 4h, and adding 50mg of HA (M is 10 mg)5) Stirring at room temperature for 12h, centrifuging at 8000rmp, collecting precipitate, washing for 3 times, and resuspending to obtain MSe-HA NPs aqueous solution.
(3) The synthesis method of nano selenium (Se NPs) comprises the following steps: 5mL of sodium selenite solution (5mg/mL) is stirred for 30min, and then 10mL of reducing agent L-ascorbic acid solution (10mg/mL) is added dropwise and stirring is continued for 2 h. And after the reaction is finished, centrifuging and washing for three times to obtain the nano-selenium.
FIG. 1 is a transmission electron micrograph and a specific surface inspection image of MSe NPs obtained in example 1, wherein A is a transmission electron micrograph of 39000 times; b is a transmission electron microscope image which is 65000 times; c is a nitrogen adsorption desorption curve chart; d is the aperture distribution diagram. As can be seen from FIGS. 1A and 1B, the MSe NPs are nanoparticles uniformly dispersed at about 150nm, and the porous structures are uniformly distributed on the surfaces of the MSe NPs, and further detection through nitrogen adsorption and desorption experiments shows that (FIGS. 1C and 1D), the MSe NPs are obvious porous structures, the pore diameters of the MSe NPs are mainly concentrated at 17.2nm, and the specific surface area is 1160.196g/m2. FIG. 2 is a transmission electron micrograph and a composite image of MSe-HA NPs obtained in example 1, wherein A is a transmission electron micrograph, B is an infrared spectrum, and C is an ultraviolet spectrum. As can be seen from the transmission electron microscope image, the nano-particle size of the porous nano-selenium after hyaluronic acid modification is increased to about 155nm, and ultraviolet rays and infrared rays show that hyaluronic acid is successfully modified on the porous nano-selenium.
Example 2: active oxygen experiments in traditional nano-selenium (Se NPs), porous selenium (MSe) and hyaluronic acid-porous nano-selenium (MSe-HA) elimination solution
In this example, MSe-HA and SenPs were prepared as described in example 1.
(1) Superoxide anion elimination experiments: 2.28mg of xanthine is dissolved in 1mL of 10mM NaOH solution to prepare a homogeneous solution with xanthine concentration of 15mM for later use. mu.L of diluted xanthine (0.6mM) solution and 4.8. mu.L of xanthine oxidase were added to 805.2. mu.L of PBS buffer and reacted at 37 ℃ for 40 min. Then 100 μ L of Se NPs, MSe and MSe-HA solution with concentration of 20 μ g/mL are added respectively for incubation for 40min, while blank control groups are set, and three repeat groups are set for each group. Then adding 50 μ L of dihydropyridine probe, incubating for 40min, and detecting fluorescence intensity of each component on computer with excitation wave of 470nm and emission wave of 650nm, the result is shown in FIG. 3.
(2) Hydroxyl radical elimination experiment: taking 18mg of FeSO4.7H2O was dissolved in 10mL of deionized water (pH 3-4), and 10. mu.L of a hydrogen peroxide solution (30%) was diluted to 5 mL. Then 4mL of hydrogen peroxide diluent was added to 4mL of FeSO4Reacting in the solution for 10min, dividing the solution into four groups after the reaction is finished,100 μ L of Se NPs, MSe and MSe-HA solutions with the concentration of 200 μ g/mL are added respectively for incubation for 1h, while blank control groups are set, and three repeat groups are set for each group. Then 1mL of salicylic acid solution (1.8mM) is added to incubate for 15min, and fluorescence intensity is detected on a computer, wherein excitation wave is 385nm and emission wave is 510nm, and the result is shown in FIG. 3.
(3) Hydrogen peroxide elimination experiment: terephthalic acid was dissolved in 10mL of N, N-Dimethylformamide (DMF) to prepare a 0.5mM solution for use. 10 μ L of a hydrogen peroxide stock solution (30%) was diluted to 5 mL. Then 4mL of hydrogen peroxide diluent is divided into four groups, 100 μ L of Se NPs, MSe and MSe-HA solution with the concentration of 200 μ g/mL are respectively added for incubation for 6h, a blank control group is simultaneously arranged, and three repeat groups are arranged in each group. Then 1mL of terephthalic acid solution is added for incubation for 30min, and then the fluorescence intensity is detected on a machine, wherein the excitation wave is 320nm, and the emission wave is 425nm, and the result is shown in figure 3.
FIG. 3 is a graph showing the effect of Se NPs, MSe NPs and MSe-HA NPs obtained in example 1 on eliminating active oxygen. The reaction of xanthine with xanthine oxidase produces superoxide anion (.O)2-, superoxide anion can oxidize a dihydroethidium chloride fluorescent probe (HE) to generate ethidium chloride with special absorption at 650nm, so that the concentration of superoxide anion in the solution is directly reflected by the fluorescence intensity. As shown in fig. 3A, after fluorescence detection and background subtraction, mean fluorescence intensities of the blank control group, Se NPs group, MSe NPs group and MSe-HA NPs group were found to be 292.1, 127.6, 69.3 and 64.9, respectively, and as shown in fig. 3D, superoxide anions of the Se NPs group, MSe NPs group and MSe-HA NPs group were eliminated at 50.17%, 78.73% and 78.81%, respectively. Therefore, it is proved that the efficiency of eliminating superoxide anion free radicals by the nano selenium is effectively improved by increasing the specific surface area.
The reaction of ferrous ions with hydrogen peroxide will generate active oxygen hydroxyl radical (. OH), which can oxidize Salicylic Acid (SA) to generate 2, 3-dihydroxybenzoic acid with special absorption at 510nm, so the fluorescence intensity directly reflects the concentration of hydroxyl radical in the solution. As shown in fig. 3B, after fluorescence detection and background subtraction, the average fluorescence absorbances of the blank control group, Se NPs group, MSe NPs group and MSe-HA NPs group were found to be 0.58368, 0.29104, 0.12391 and 0.1002, respectively. As shown in FIG. 3E, the hydroxyl radical elimination efficiency of the Se NPs group, the MSe NPs group and the MSe-HA NPs group was 55.92%, 78.91% and 82.83%, respectively. Therefore, it is proved that the efficiency of eliminating hydroxyl radicals by the nano selenium is effectively improved by increasing the specific surface area.
The reaction of terephthalic acid with hydrogen peroxide results from 2-hydroxy terephthalic acid having a specific absorption at 425nm, so the magnitude of the fluorescence intensity directly reflects the concentration of hydrogen peroxide in solution. As shown in FIG. 3C, the average fluorescence absorbances of the blank control group, the Se NPs group, the MSe NPs group and the MSe-HA NPs group were found to be 1746, 1337, 219.3 and 179.8, respectively, by fluorescence detection after subtracting the background. As shown in FIG. 3F, the hydroxyl radical elimination efficiency of the Se NPs group, the MSe NPs group and the MSe-HA NPs group was 32.12%, 87.32% and 89.81%, respectively. Therefore, it is proved that the efficiency of eliminating hydrogen peroxide by nano selenium is effectively improved by increasing the specific surface area.
Example 3: porous selenium, hyaluronic acid-porous nano-selenium targeting experiment
(1) RAW264.7 cell uptake experiments: to test the targeting ability of the porous nano-selenium (MSe NPs) and the hyaluronic acid-porous nano-selenium (MSe-HA NPs) obtained in example 1, we tested the uptake ability of LPS-activated RAW264.7 cells to MSe NPs and MSe-HA NPs using confocal laser microscopy. Briefly, activated RAW264.7 cells were treated at 1X 105Density in confocal dishes and incubation for 24h, followed by addition of 50 μ L of medium containing FITC-labeled MSe NPs (10 μ g/mL) and MSe-HA NPs (10 μ g/mL) to the cells, respectively, after incubation of the cells with the drug for 0.5h, 1h, 2h, 4h and 8h, respectively, the cells were washed three times with PBS and stained with DAPI for 15 min. The cell samples were washed again with PBS and observed under confocal laser microscopy, and the results are shown in FIG. 4.
(2) In vivo targeting experiments of inflammatory mice: in order to detect the inflammatory site targeting ability of the porous nano-selenium (MSe NPs) and the hyaluronic acid-porous nano-selenium (MSe-HA NPs), the accumulation amount of the MSe NPs and MSe-HA NPs at the inflammatory site is observed by fluorescence live imaging, and the stronger the fluorescence intensity, the more the accumulation amount of the nano-selenium is, the stronger the targeting ability is. Briefly, 10 μ L of Lipopolysaccharide (LPS) solution at a concentration of 10ng/mL was injected into the right ear of C57BL/6, after 12h, a significant red swelling of the ear indicated a significant inflammatory response in the right ear, followed by injection of PBS aqueous solution containing FITC-labeled MSe NPs (10 μ g/mL) and MSe-HA NPs (10 μ g/mL) into mice via the tail vein at an injection rate of 200 μ g/kg (drug/mouse body weight), followed by observation of the distribution of in vivo fluorescence at 0.5h, 1h, 2h, 4h, 8h and 12h, respectively, and the results are shown in FIG. 4.
FIG. 4 is a map of the MSe NPs and MSe-HA NPs obtained in example 1, in macrophage cells and mice, wherein A is the cellular uptake pattern of macrophages; b is a fluorescence quantification graph in the cell absorption of macrophages; c is a porous nano-selenium in vivo targeting map; d is a target fluorescence quantification picture in the body of the porous nano selenium.
Confocal laser detection shows that the fluorescence intensity of MSe and MSe-HA cells and RAW264.7 cells is time-dependent within 2h of incubation. And the fluorescence intensity reached saturation after 2h without significant enhancement. In addition, the fluorescence intensity of MSe-HA at 2h is obviously stronger than MSe through comparison, and the fluorescence semiquantitative display shows that the fluorescence intensity of MSe-HA is 1.98 times of MSe (FIG. 4B), so absorption experiments show that the target of the porous nano selenium to macrophages can be enhanced through hyaluronic acid modification. As shown in FIG. 4C, the results of in vivo fluorescence experiments further showed that MSe-HA reached the site of inflammation at 1h and accumulated efficiently. And the fluorescence intensity reaches the maximum at 4h and is 4 times of MSe (fig. 4D), and the fact that the hyaluronic acid modified porous nano selenium has excellent targeting property on inflammation is also proved.
Example 4: experiment for eliminating active oxygen in cells by using traditional nano-selenium, porous selenium and hyaluronic acid-porous nano-selenium
Se NPs, MSe NPs and MSe-HA NPs in this example were prepared as described in example 1.
In order to detect the capacity of eliminating and activating active oxygen in macrophages of traditional nano-selenium (Se NPs), porous nano-selenium (MSe NPs) and hyaluronic acid-porous nano-selenium (MSe-HA NPs), a flow cytometer and a confocal laser microscope are adopted to detect that RAW264.7 cells pass through various cellsThe fluorescence intensity of intracellular reactive oxygen species after treatment. Briefly, activated RAW264.7 cells were treated at 1X 105The cells were seeded in a confocal dish and incubated for 24h, then 50. mu.L of a medium containing Se NPs, MSe NPs and MSe-HA NPs at a concentration of 10. mu.g/mL were added to the cells, respectively, while a blank control was set, and after incubating the cells with the drug for 12h, the cells were washed three times with PBS and stained with a reactive oxygen species probe (DCFH-DA) for 30 min. The cell samples were washed again with PBS and observed under confocal laser microscopy (excitation and emission wavelengths of 495nm and 525nm, respectively). The flow cytometry detection is to detect the activated RAW264.7 cells at 5X 10 per hole4The density is in six-hole plate, and the temperature is 37 ℃, 5% CO2After 24h of incubation in an incubator, 50. mu.L of media containing Se NPs, MSe NPs and MSe-HA NPs at a concentration of 10. mu.g/mL were added to the cells, while a blank control was set, and after 12h of incubation of the cells with the drug, the cells were washed three times with PBS and stained with active oxygen probe (DCFH-DA) for 30 min. The cell samples were washed again with PBS and examined in a flow cytometer, and the results are shown in figure 5.
FIG. 5 is a graph showing the results of experiments on the elimination of activated oxygen in macrophages by Se NPs, MSe NPs and MSe-HA NPs obtained in example 1. Laser confocal detection shows that after incubation for 12 hours with RAW264.7 induced by LPS, as shown in fig. 5A, the fluorescence intensity of active oxygen in the blank control group (LPS) is the strongest, while the fluorescence intensity of active oxygen in cells in each experimental group added with nano-action shows reduction in different degrees, and it can be found that the efficiency of MSe and MSe-HA for eliminating active oxygen is significantly stronger than that of the traditional nano-selenium (Se), thereby also proving that the nano-selenium with a porous structure HAs stronger active oxygen elimination capability. Moreover, MSe-HA HAs better targeting ability, and its efficiency of eliminating active oxygen is stronger than MSe, and the active oxygen in cell is eliminated basically and completely after treatment. The flow cytometry detection shows the same trend, as shown in fig. 5B, the result detection shows that the fluorescence intensity of the active oxygen in the blank control group is 2315, the fluorescence intensity of the active oxygen in the cells after the Se NPs treatment is 1238, and the fluorescence intensity after the MSe and MSe-HA treatment is only 900 and 804, so the MSe-HA with the targeting ultra-large specific surface HAs good macrophage active oxygen elimination capability.
Example 5: experiment for eliminating in vivo active oxygen by using traditional nano selenium, porous selenium and hyaluronic acid-porous nano selenium to treat septicemia
Se NPs, MSe NPs and MSe-HA NPs in this example were prepared as in example 1.
C57BL/6 mice with the average weight of about 20.0g and the age of 6-8 weeks are averagely divided into four groups, 5 mice in each group are molded, 10 mu L of physiological saline solution (100ng/mL) dissolved with lipopolysaccharide is injected into the right ear of the mouse, and the treatment is carried out after 12h when the right ear of the mouse is inflamed. Namely, PBS aqueous solutions of Se NPs, MSe NPs and MSe-HA NPs with a concentration of 2mg/mL were injected into tail vein respectively, and the injection amount was 200. mu.g/Kg (drug/mouse body weight), while a blank control group was set. The red and swollen condition of the right ear of the mouse is observed every 24h and recorded by taking a picture by a mobile phone. Four days after treatment, active oxygen probe (DCFH-DA) was injected and the concentration of active oxygen in the right ear of the mouse was observed using in vivo fluorescence imaging. And simultaneously, observing the septicemia treatment condition by observing the liver and lung organ injury slice. The results are shown in FIG. 6.
FIG. 6 is a graph showing that Se NPs, MSe NPs and MSe-HA NPs obtained in example 1 can eliminate reactive oxygen species in mice to treat septicemia. As shown in FIG. 6A, the right ear of the mice in the blank control group was significantly damaged at the fourth day, which is the oxidative damage caused by the high concentration of active oxygen at the inflammatory site in the control group. The treatment groups injected with Se NPs, MSe NPs and MSe-HA NPs show different degrees of treatment effects, wherein the MSe NPs are slightly red and swollen, and no damage occurs, and MSe-HA NPs completely inhibit the damage, so that the damage is the same as that of normal mice. As shown in FIG. 6B, in vivo imaging examination further showed that the concentration of active oxygen was significantly higher in the control group than in the treated group, active oxygen was completely inhibited in the MSe NPs and MSe-HA NPs treated group, and complete hepatopulmonary injury was observed in the MSe NPs and MSe-HA NPs treated group from the hepatopulmonary section (FIG. 6D). These results indicate that MSe NPs and MSe-HA NPs also have good active oxygen elimination ability in vivo to effectively inhibit inflammatory oxidative damage and treat sepsis.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. The preparation method of the hyaluronic acid-porous nano selenium compound is characterized by comprising the following steps:
mixing the porous nano-selenium aqueous solution with EDC and NHS, activating, and mixing and reacting the activated porous nano-selenium aqueous solution with hyaluronic acid to obtain a hyaluronic acid-porous nano-selenium compound aqueous solution;
the preparation method of the porous nano selenium aqueous solution comprises the following steps:
dissolving mesoporous silica powder in an organic solvent, mixing with APTES, performing reflux reaction to obtain an aminated mesoporous silica solution, then dropwise adding a sodium selenite aqueous solution, stirring for reaction, dropwise adding a reducing agent, continuously stirring for reaction to obtain a reaction solution after the reaction is finished, and then adding hydrofluoric acid to etch away the mesoporous silica to obtain the porous nano-selenium.
2. The method of preparing a hyaluronic acid-porous nano-selenium complex according to claim 1, characterized in that:
the concentration of the porous nano-selenium in the porous nano-selenium aqueous solution is 0.1-10 mg/mL;
the mol ratio of the porous nano selenium to the EDC to the NHS is 1: 0.1: 0.1-1: 20: 20;
the molecular weight of the hyaluronic acid is 10 KD-50 KD;
the mass ratio of the porous nano selenium to the hyaluronic acid is 1: 1-1: 10.
3. the method of preparing a hyaluronic acid-porous nano-selenium complex according to claim 1, characterized in that:
the activation time is 0.5-10 h;
the mixing reaction condition is stirring reaction at room temperature for 6-18 h;
and after the mixing reaction is finished, the purification process is also included, specifically, the solution after the mixing reaction is centrifugally collected, precipitated and washed, and then is resuspended, so that the hyaluronic acid-porous nano selenium compound aqueous solution is obtained.
4. The method of preparing a hyaluronic acid-porous nano-selenium complex according to claim 1, characterized in that:
the mass-volume ratio of the mesoporous silica to the organic solvent to the APTES is 0.1 g: 5mL of: 10 mu L-20 g: 100mL of: 100 mu L of the solution;
the volume ratio of the aminated mesoporous silica solution to sodium selenite is 1: 2-1: 10;
the molar ratio of the sodium selenite to the reducing agent is 1: 1-1: 5;
the time of the first stirring reaction is 15-60 min, and the time of the second stirring reaction is 1-5 h.
5. The method of preparing a hyaluronic acid-porous nano-selenium complex according to claim 1, characterized in that:
the concentration of sodium selenite in the sodium selenite aqueous solution is 0.1-1.0 mol/L;
the reducing agent is at least one of citric acid, aspartic acid, glutamic acid, retinoic acid and L-ascorbic acid;
the concentration of the hydrofluoric acid is 20-70 wt%.
6. The method for preparing the hyaluronic acid-porous nano selenium compound of claim 1, wherein the mesoporous silica is prepared by the following steps:
dissolving cetyl trimethyl ammonium bromide in water, adding an organic solvent A, then adjusting the solution to be alkaline, adding tetraethoxysilane after stirring and dissolving, raising the temperature, stirring and reacting to obtain a reaction solution, and then refluxing the reaction solution in a mixed solution of an acidic solution and an organic solvent B to obtain the mesoporous silicon dioxide.
7. The method of preparing a hyaluronic acid-porous nano-selenium complex according to claim 6, wherein:
the mass volume ratio of the hexadecyl trimethyl ammonium bromide to the water to the organic solvent A is 50 mg: 20mL of: 5 mL-200 mg: 100mL of: 50 mL;
the molar ratio of the ethyl orthosilicate to the hexadecyl trimethyl ammonium bromide is 1: 1-1: 10;
the temperature is raised to 45-70 ℃ and then the stirring reaction is continued for 12-36 h;
the refluxing time is 8-12 h.
8. A hyaluronic acid-porous nano selenium complex prepared according to the method of any of claims 1 to 7.
9. Use of the hyaluronic acid-porous nanosele complex according to claim 8 for the preparation of a medicament for the treatment of sepsis.
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