CN114288319A - Application of functionalized nano-selenium in preparation of anti-wound infection medicine - Google Patents
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Abstract
The invention discloses application of functionalized nano-selenium in preparation of a medicament for resisting wound infection, wherein the functionalized nano-selenium is more than one of polymer modified nano-selenium such as chitosan nano-selenium, polyvinylpyrrolidone nano-selenium or lentinan nano-selenium. The invention synthesizes nano-selenium with different charge modifiers, which not only can realize the direct killing effect on pathogenic bacteria, but also can lead immune cells to effectively eliminate pathogenic bacteria infection by regulating and controlling the immune system of an organism. The functional nano-selenium has positive and effective regulation and control effects on immune cells at a wound part, has good immune activation capability, promotes maturation and differentiation of DC cells and NK cells, promotes phagocytosis of bacteria by macrophages, activates immune response, and quickly sterilizes bacteria, thereby accelerating repair of the wound.
Description
Technical Field
The invention relates to application of functionalized nano-selenium in preparation of a medicament for resisting wound infection.
Background
A large number of studies indicate that the main pathogenic bacteria of wound infection include gram-negative bacteria such as escherichia coli, pseudomonas aeruginosa and the like, and gram-positive bacteria such as staphylococcus aureus, coagulase-negative staphylococcus and the like, and the bacteria generate drug resistance to broad-spectrum antibiotics. Infection with multidrug resistant methicillin-resistant staphylococcus aureus (MRSA) has become a global treatment problem, and the lowest inhibitory concentration is the index commonly used in clinical practice to evaluate the antibacterial activity of antibacterial agents.
At present, the prevention and treatment measures for drug-resistant bacteria wound infection mainly comprise antibacterial drug combination, research and development of novel antibacterial drugs (antibacterial agents such as nano silver and the like), and the like. However, these strategies have their limitations, for example, combination of drugs, while effective in improving antibacterial effect, also greatly increase the risk of multiple drug resistance by bacteria; although nano silver shows good antibacterial activity, the potential safety problem is difficult to solve, and the wide clinical application of nano silver is greatly limited.
Serious trauma can also induce problems such as dysfunction of the immune system of the body. In recent years, immunotherapy has shown good application prospects in severe wound treatment. The immune system of an organism is remodeled mainly by using an immune activator or a cytokine, such as gamma-interferon (IFN-gamma), granulocyte-macrophage colony stimulating factor (GM-CSF) and the like, so that pathogenic bacteria are effectively killed by immune cells, and the risk of wound infection is greatly reduced. However, the prolonged release of higher levels of proinflammatory cytokines by immune cells is detrimental to wound healing. Therefore, the development of a safe, effective, broad-spectrum new antibiotic or antibacterial drug has been a key problem to be solved urgently in this field.
Disclosure of Invention
The invention aims to provide application of functionalized nano-selenium in preparation of a medicament for resisting wound infection, wherein the functionalized nano-selenium has a direct antibacterial effect and an immune regulation function.
The purpose of the invention is realized by the following technical scheme:
the application of the functionalized nano-selenium in preparing the anti-wound infection medicine;
the functional nano selenium is more than one of chitosan nano selenium (CS-SeNPs), polyvinylpyrrolidone nano selenium (PVP-SeNPs), lentinan nano selenium (LNT-SeNPs), oyster mushroom polysaccharide nano selenium (PTR-SeNPs), Tween 80 nano selenium (TW80-SeNPs), polyethylene glycol nano selenium (PEG-SeNPs) or polyallylamine nano selenium (PAH-SeNPs); the functionalized nano-selenium has a certain immunostimulation function, presents a concentration-dependent effect and has a direct killing or inhibiting effect on pathogenic bacteria.
The chitosan nano selenium (CS-SeNPs) and the preparation method thereof have been disclosed in the prior art, such as Chinese patent application CN 112870348A, Chinese patent application CN 109588235A and Chinese invention patent CN 106692181B;
preferably, the chitosan nano selenium is prepared by the following steps:
mixing chitosan solution with sodium selenite (Na)2SeO3) Uniformly mixing the solution, dropwise adding a reducing agent solution, reacting overnight at room temperature, dialyzing the reaction solution to remove unreacted polymers, and thus obtaining chitosan nano selenium;
the concentration of the chitosan solution is 0.1-20 mg/ml; preferably 5 mg/ml.
The concentration of the sodium selenite solution is 0.5-200 mM; preferably 100 mM.
The concentration of the reducing agent solution is 2-800 mM; preferably 400 mM.
The polyvinylpyrrolidone nano selenium (PVP-SeNPs) and the preparation method thereof are disclosed in Chinese invention patent CN 106692181B;
preferably, the polyvinylpyrrolidone nano-selenium is prepared by the following steps:
mixing polyvinylpyrrolidone solution with sodium selenite (Na)2SeO3) The solution is mixed evenly and drippedAdding a reducing agent solution, reacting at room temperature overnight, dialyzing the reaction solution to remove unreacted polymers, and preparing polyvinylpyrrolidone nano selenium;
the concentration of the polyvinylpyrrolidone solution is 0.1-20 mg/ml; preferably 10 mg/ml.
The concentration of the sodium selenite solution is 0.5-200 mM; preferably 100 mM.
The concentration of the reducing agent solution is 2-800 mM; preferably 400 mM.
The lentinan nano selenium (LET-SeNPs) and the preparation method thereof are disclosed in Chinese invention patent applications CN 109588235A and CN 112999241A;
preferably, the lentinan nano-selenium is prepared by the following steps:
mixing lentinan solution with sodium selenite (Na)2SeO3) Uniformly mixing the solution, dropwise adding a reducing agent solution, reacting at room temperature overnight, dialyzing the reaction solution to remove unreacted polymers, and thus obtaining lentinan nano selenium;
the concentration of the lentinan solution is 0.1-20 mg/ml; preferably 10 mg/ml.
The concentration of the sodium selenite solution is 0.5-200 mM; preferably 100 mM.
The concentration of the reducing agent solution is 2-800 mM; preferably 400 mM.
The molar ratio of the sodium selenite to the reducing agent is 1: 1-1: 8; preferably 1: 4.
The reducing agent is vitamin C (Vc), hydrazine hydrate or citric acid.
The dialysis is preferably carried out for 12-24h by using a 10000-100000kDa dialysis bag.
The oyster mushroom polysaccharide nano selenium (PTR-SeNPs), Tween 80 nano selenium (TW80-SeNPs), polyethylene glycol nano selenium (PEG-SeNPs) or polyacrylamide nano selenium (PAH-SeNPs) and the preparation method thereof are disclosed in the prior art, such as Liu T, Xu L, He L, ZHao J, Zhang Z, Chen Q, Chen T.2020.Selenium nanoparticies regulation styrene to ethylene-induced yeast cells-based cultured microorganism.
Yang Y,Zhang Z,Chen Q,You Y,Li X,Chen T.2021.Functionalized Selenium Nanoparticles Synergizes With Metformin to Treat Breast Cancer Cells Through Regulation of Selenoproteins.Front Bioeng Biotechnol 9:758482.
Zeng D,Zhao J,Luk KH,Cheung ST,Wong KH,Chen T.2019.Potentiation of in Vivo Anticancer Efficacy of Selenium Nanoparticles by Mushroom Polysaccharides Surface Decoration.J Agric Food Chem 67:2865-2876.
Compared with the prior art, the invention has the following advantages and effects:
the invention synthesizes nano-selenium with different charge modifiers, which not only can realize the direct killing effect on pathogenic bacteria, but also can lead immune cells to effectively eliminate pathogenic bacteria infection by regulating and controlling the immune system of an organism. The functional nano-selenium has positive and effective regulation and control effects on immune cells at a wound part, has good immune activation capability, promotes maturation and differentiation of DC cells and NK cells, promotes phagocytosis of bacteria by macrophages, activates immune response, and quickly sterilizes bacteria, thereby accelerating repair of the wound.
Drawings
FIG. 1 shows the bacteriostatic effect of different concentrations of functionalized nano-selenium on methicillin-resistant Staphylococcus aureus (MRSA) plates.
FIG. 2 is the effect of functionalized nano-selenium on dendritic cell maturation.
Figure 3 is a quantification of the effect of functionalized nano-selenium on dendritic cell maturation.
FIG. 4 effect of functionalized nano-selenium on macrophage phagocytosis of bacteria.
FIG. 5 is a quantification of the effect of functionalized nano-selenium on the expression of NKG2D receptor on the surface of natural killer cells.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
Synthesis of functionalized nano-selenium
(1) 100mg of CS (chitosan, Mecanne) and LNT (lentinan, Shaanxi) are takenForest biotechnology limited), PVP (polyvinylpyrrolidone, mclin) is dissolved in 10mL of deionized water respectively to prepare stock solutions with the concentration of 10 mg/mL; 173mg of Na are taken2SeO3Dissolving sodium selenite (Acfaesar) and Vc (ascorbic acid, Meclin) 176mg in 10mL of deionized water respectively to prepare stock solutions with the concentration of 100 mM;
(2) 2mL of CS solution was taken and 0.5mL of Na was added2SeO3Adding deionized water to 8mL of the solution, and stirring the solution on a magnetic stirrer for 5min to ensure that the polymer is mixed with Na2SeO3And (3) fully mixing to form a mixed solution, and then dropwise adding 2mL of Vc solution into the mixed solution until the Vc is dropwise added, and stirring at room temperature overnight. Putting the reacted solution into a 100000kDa dialysis bag, dialyzing for 24h, and removing unreacted polymers to obtain chitosan nano selenium;
the preparation method of the polyvinylpyrrolidone nano-selenium and the lentinan nano-selenium is the same as that of the chitosan nano-selenium, and comprises the use amount and the proportion of raw materials.
Example 2
Bacteriostatic experiment of functionalized nano-selenium
Methicillin-resistant staphylococcus aureus (ATCC43300) was selected and inoculated into LB medium and cultured overnight on a constant temperature shaker at 37 ℃. The bacteria were then diluted to a concentration of 10 with LB medium7CFU mL-1. Diluted bacteria (5. mu.L, 10) were added to each ep tube7CFU mL-1). PBS buffer (0.01mmol/L, pH 7.4, as a blank control), chitosan nano-selenium (CS-SeNPs), polyvinylpyrrolidone nano-selenium (PVP-SeNPs), lentinan nano-selenium (LNT-SeNPs) were then diluted to 500 μ L in ep tubes, respectively, and incubated on a shaking shaker at 37 ℃ overnight. Diluting by a certain multiple, and demonstrating the antibacterial effect through a plate coating experiment.
As shown in FIG. 1 and Table 1, the antibacterial effect was strong against MRSA, LNT-SeNPs, with a Minimum Inhibitory Concentration (MIC) of 8ug/ml and a Minimum Bactericidal Concentration (MBC) of 64 ug/ml; the PVP-SeNPs also have certain antibacterial effect, the Minimum Inhibitory Concentration (MIC) is 8ug/ml, and the Minimum Bactericidal Concentration (MBC) is 128 ug/ml; CS-SeNPs exhibit poor antibacterial activity because of their instability in bacterial LB medium, and are suspected to be highly affected by external pH and to be prone to coagulation.
TABLE 1
| Unit: mu.g/ml | CS-Se NPs | PVP-Se NPs | LET- |
| MIC | |||
| 64 | 8 | 8 | |
| |
128 | 128 | 64 |
Example 3
Immune activation effect experiment of functional nano-selenium
(1) Effect of functionalized nano-selenium on dendritic cell maturation
DC cells have an important biological property, namely the maturation of DC cells. DC cells are first stimulated by pathogens or other antigens, and then take up and process the antigens, gradually differentiating into mature cells. Then, the DC cells migrate from the peripheral tissues to lymph nodes, spleen, and the like, and present antigens to T cells, thereby initiating an immune response and exerting a bactericidal effect.
In the experiment, female C57BL/6 mice are used, after being sacrificed, bone marrow of thighbone and shinbone of the mice is collected aseptically, and after red blood cells are cracked, single cell suspension is obtained; then, the cells were cultured in a culture medium containing GM-CSF cytokine for 6 to 8 days in a carbon dioxide cell incubator to obtain bone marrow-derived dendritic cells (BMDCs). Then, different functionalized nano-selenium (selenium concentration is 1.5 mug/mL) and BMDC cells are respectively incubated together overnight, and Lipopolysaccharide (LPS) is used as a positive control group; finally, the cells are harvested and BMDC cell maturation is detected using flow cytometry using a variety of fluorescently labeled antibodies such as FITC-CD11c, PE-CD80, and APC-CD 86. As shown in fig. 2 and 3, the positive control Lipopolysaccharide (LPS) -treated DC cells matured 94.93%, and the blank control DC cells matured 36.29%. The maturity of DC cells treated by naked selenium (SeNPs) is improved to 51.60%, and the maturity of the DC cells treated by the functionalized nano selenium PVP-SeNPs, CS-SeNP and LNT-SeNPs exceeds 60%. Therefore, the nano-selenium has a certain effect on DC activation, wherein the stimulation effect of the nano-selenium modified by PVP, CS and LNT is obviously enhanced.
(2) Effect of functionalized nano-selenium on phagocytosis of bacteria by macrophages
Macrophages are important components of the body's innate immunity, and also are the primary effector cells that initiate specific immune responses through antigen presentation, recognize, phagocytize cells and digest pathogens, and also can ingest extracellular substances through pinocytosis and receptor-mediated endocytosis. The enhancement of the endocytosis capacity is beneficial to promoting the enhancement of the immunopotency of the macrophage, and the influence of the functionalized nano selenium on the endocytosis of the macrophage is not reported at present.
1ml methicillin-resistant Staphylococcus aureus (1X 10)8CFU/ml) was dark stained with 10. mu.M 5(6) -carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, 100. mu.g/ml) for 30 min. Subsequently, the stained broth was centrifuged to remove CFDA-SE and rinsed with PBS. Macrophage (2X 10) incubation with different functionalized nano-selenium (selenium concentration 1. mu.g/mL)5/ml)24 hours, then scraping the cells, and mixing with 1X 108CFU/ml of 100. mu.l stained MRSA cocktail90min (gently rinse every 30 min). The cells were then centrifuged to remove non-phagocytic bacteria from the supernatant and the cells were washed with PBS and analyzed on a flow cytometer.
Results shown in FIG. 4, PVP-SeNPs, CS-SeNP, LNT-SeNPs and naked selenium all have certain effects on increasing phagocytic bacterial functions of macrophages, and the effects are improved from 42% to more than 60%, wherein the stimulation effects of different nano selenium are not greatly different.
(3) Effect of functionalized nano-selenium on activation of natural killer cells
In recent years, research on anti-infection immunity related to Natural Killer (NK) cells has been greatly advanced. There is increasing evidence that NK cells play an important role in the infection by a variety of pathogens, including viruses, bacteria, fungi and protozoa. The NK cell surface has NKG2D receptor, and its ligand is NKG2DL, which is the sign of NK cell activation.
NK cells were cultured in a petri dish for 24 hours, and then incubated for 24 hours with a medium containing 1.5. mu.g/mL of SeNPs, PVP-SeNPs, CS-SeNPs, and LNT-SeNPs.
Cells were diluted to about 10 ten thousand/mL with PBS. Mu.l of the above cell suspension was added to each tube, 10. mu.g/mL of FITC-anti-NKG2D was added, and the mixture was incubated at 4 ℃ for 30 min. Cells were washed twice with PBS and centrifuged at 1000rpm/min for 5min each time. The cells were resuspended in PBS and analyzed on a flow cytometer.
The results are shown in FIG. 5, PVP-SeNPs, CS-SeNPs and LNT-SeNPs and polymer-free polysaccharide-modified SeNPs have significant effects on activating NK cells, wherein the nano-selenium activating effect after modification by PVP, CS and LNT is obviously enhanced, and the LNT-SeNPs activating effect is strongest.
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 (10)
1. The application of the functionalized nano-selenium in preparing the anti-wound infection medicine is characterized in that: the functional nano-selenium is one or more of polymers or polysaccharide modified nano-selenium such as chitosan nano-selenium, polyvinylpyrrolidone nano-selenium, lentinan nano-selenium, oyster mushroom polysaccharide nano-selenium, tween 80 nano-selenium, polyethylene glycol nano-selenium, polyacrylamide nano-selenium and the like.
2. Use according to claim 1, characterized in that: the chitosan nano selenium is prepared by the following steps: uniformly mixing the chitosan solution and the sodium selenite solution, dropwise adding a reducing agent solution, reacting at room temperature overnight, dialyzing the reaction solution to remove unreacted polymers, and thus obtaining the chitosan nano-selenium.
3. Use according to claim 2, characterized in that: the concentration of the chitosan solution is 0.1-20 mg/ml.
4. Use according to claim 2, characterized in that:
the concentration of the sodium selenite solution is 0.5-200 mM;
the concentration of the reducing agent solution is 2-800 mM.
5. Use according to claim 1, characterized in that: the polyvinylpyrrolidone nano selenium is prepared by the following steps: uniformly mixing a polyvinylpyrrolidone solution and a sodium selenite solution, dropwise adding a reducing agent solution, reacting at room temperature overnight, dialyzing the reaction solution to remove unreacted polymers, and thus obtaining the polyvinylpyrrolidone nano-selenium.
6. Use according to claim 5, characterized in that: the concentration of the polyvinylpyrrolidone solution is 0.1-20 mg/ml.
7. Use according to claim 5, characterized in that:
the concentration of the sodium selenite solution is 0.5-200 mM;
the concentration of the reducing agent solution is 2-800 mM.
8. Use according to claim 1, characterized in that: the lentinan nano selenium is prepared by the following steps: uniformly mixing the lentinan solution and the sodium selenite solution, dropwise adding a reducing agent solution, reacting at room temperature overnight, dialyzing the reaction solution to remove unreacted polymers, and thus obtaining the lentinan nano-selenium.
9. Use according to claim 8, characterized in that: the concentration of the lentinan solution is 0.1-20 mg/ml.
10. Use according to claim 8, characterized in that:
the concentration of the sodium selenite solution is 0.5-200 mM;
the concentration of the reducing agent solution is 2-800 mM.
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| CN104447660A (en) * | 2014-11-24 | 2015-03-25 | 暨南大学 | Functional nano-selenium and preparation thereof as well as application of functional nano-selenium in preparation of anti-bacteria and sterilization drugs |
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| CN104447660A (en) * | 2014-11-24 | 2015-03-25 | 暨南大学 | Functional nano-selenium and preparation thereof as well as application of functional nano-selenium in preparation of anti-bacteria and sterilization drugs |
Non-Patent Citations (2)
| Title |
|---|
| ABDOLRASOUL RANGRAZI等: "Synthesis and antibacterial activity of colloidal selenium nanoparticles in chitosan solution: a new antibacterial agent" * |
| 王超等: "纳米硒复合物的研究进展" * |
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