CN112704735B - Inorganic ion mediated organic compound nano enzyme, preparation method and application - Google Patents
Inorganic ion mediated organic compound nano enzyme, preparation method and application Download PDFInfo
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Abstract
The invention belongs to the field of materials, and particularly relates to an inorganic ion-mediated organic compound nanoenzyme, and a preparation method and application thereof. The invention provides a nano enzyme with inorganic ion mediation, which is prepared by the following steps: dissolving the compound M1 and inorganic ions in tetrahydrofuran, stirring at room temperature, and uniformly mixing to obtain a compound M1-Fe; dissolving a compound M1-Fe and polylactic acid-glycolic acid copolymer-polyethylene glycol (PLGA-PEG) in tetrahydrofuran, adding into ultrapure water of a filter membrane under an ultrasonic condition, and performing ultrasonic treatment for 5-8 min to obtain the nano enzyme. The inorganic ions not only enhance the photo-thermal property of the compound, but also add H to the formed nano enzyme2O2OH is produced. Under the condition of illumination, the nano enzyme generates local heat, can not only damage bacteria, but also accelerate the generation of OH, and realizes the photo-thermal and chemical-kinetic combined treatment.
Description
Technical Field
The invention belongs to the field of materials, and particularly relates to an inorganic ion-mediated organic compound nanoenzyme, and a preparation method and application thereof.
Background
Over the past several decades, the increasing number of diseases caused by bacteria has become one of the biggest problems that continue to threaten human health worldwide. Many traditional anti-infective treatments that rely on antibiotics are gradually becoming ineffective due to the emergence of antibiotic-resistant strains. At present, new antibacterial methods based on nanomaterials are emerging, such as photothermal therapy (PTT), photodynamic therapy (PDT) and ultrasound therapy. Wherein the photothermal therapy (PTT) has woundThe nano material with the photo-thermal property can convert light energy into heat energy, and the heat energy kills cells by destroying cell membranes and protein denaturation, thereby causing bacterial death. Compared to traditional antibiotic therapy, chemokinetic therapy (CDT) is an emerging therapeutic approach that catalyzes hydrogen peroxide (H) based on the fenton or fenton-like reaction2O2) Hydroxyl free radicals (. OH) are produced, thereby disrupting the normal physiological functions of the bacteria and inducing bacterial death. H is to be2O2This class of materials that are converted to OH is defined as nanoenzymes. And H2O2In contrast, OH is one of the strongest oxidants, with more effective antimicrobial properties. And the material with the photo-thermal property can promote the generation of OH under the condition of illumination. At present, the synergistic treatment mode is used for replacing a single treatment mode, so that the chemodynamic therapy can be activated efficiently, and the treatment effect of the wound infection caused by bacteria is promoted. The invention aims to combine photothermal therapy and chemokinetic therapy to achieve the aim of synergistically and efficiently treating bacterial infection.
Disclosure of Invention
The invention aims to provide an organic compound nano enzyme mediated by inorganic ions, which utilizes coordination of inorganic ions and organic compounds to enhance the photo-thermal property of the compounds, and the formed nano enzyme is added with H2O2Then generates OH, and simultaneously, the generation of OH is promoted by heat energy, and the treatment of bacterial infection is realized by the photothermal and chemokinetic combined therapy.
In order to achieve the purpose, the invention adopts the following technical scheme:
an inorganic ion-mediated organic compound nanoenzyme, which is prepared by the following steps:
and 2, dissolving the compound M1-Fe and polylactic acid-glycolic acid copolymer-polyethylene glycol (PLGA-PEG) in tetrahydrofuran, adding the mixture into ultrapure water of a filter membrane under an ultrasonic condition, and performing ultrasonic treatment for 5-8 min to obtain the nano enzyme.
The inorganic ion is Fe2+Said compound M1 is:
further, the molar ratio of the compound M1 to the inorganic ions in the step 1 is 1: 1-3, and the stirring time at room temperature is 2-4 h.
Further, the mass ratio of M1-Fe to polylactic acid-glycolic acid copolymer-polyethylene glycol in the step 2 is 1: 4-8.
A preparation method of inorganic ion mediated organic compound nano enzyme comprises the following steps:
the inorganic ion is Fe2+Said compound M1 is:
further, the molar ratio of the compound M1 to the inorganic ions in the step 1 is 1: 1-3, and the stirring time at room temperature is 2-4 h.
Further, the mass ratio of M1-Fe to polylactic acid-glycolic acid copolymer-polyethylene glycol in the step 2 is 1: 4-8.
Application of inorganic ion-mediated organic compound nanoenzyme for generating hydroxyl radicals by adding hydrogen peroxide, wherein the nanoenzyme is prepared by Fe2+Mediation increased the photothermal properties of M1.
Compared with the prior art, the invention has the following advantages:
the inorganic ion mediated organic compound nano enzyme obtained by the invention not only has stronger photo-thermal property than the original compound M1, but also is added with H2O2The nano enzyme can generate OH, under the condition of illumination, the nano enzyme can generate local heat, not only can damage bacteria, but also can accelerate the generation of OH, the oxidizing capability of OH is very strong, and the nano enzyme can kill bacteria or cancer cells, thereby realizing the combined efficient treatment of photo-thermal and chemical kinetics. The nano enzyme can be applied to the field of biological medicine.
Drawings
FIG. 1 is a Scanning Electron Microscope (SEM) image of the nanoenzyme of the present invention;
FIG. 2 is a normalized ultraviolet absorption spectrum of nanoenzyme (M1-FeNPs) and M1NPs of the present invention;
FIG. 3 is a test of the ability of nanoenzymes of the invention to generate hydroxyl radicals (. OH);
FIG. 4 is a temperature rise curve of the nanoenzyme (M1-FeNPs) of the present invention under 660nm laser irradiation at different concentrations;
FIG. 5 is the photothermal lift curves of the nanoenzyme (M1-FeNPs) and M1NPs of the present invention under the laser irradiation with 660nm wavelength;
FIG. 6 is a graph showing the photostability of nanoenzymes (M1-FeNPs) according to the present invention under laser irradiation at a wavelength of 660 nm;
FIG. 7 is a graph showing the bactericidal activity of the present invention containing nanoenzymes (M1-FeNPs) at pH 6.5 and pH 7.4 against Staphylococcus aureus (irradiation with laser light at 660nm, 1W/cm)2)。
Detailed Description
Example 1
A preparation method of inorganic ion mediated organic compound nano enzyme comprises the following steps:
and 2, adding the uniformly mixed compound M1-Fe and 62.5 mu L of PLGA-PEGTHF solution of 8mg/mL into THF, wherein the final volume is 4mL, adding the mixture into 10mL of ultrapure water of a filter membrane under the ultrasonic condition, and carrying out ultrasonic treatment for 5min to obtain the nano enzyme.
Example 2
A preparation method of inorganic ion mediated organic compound nano enzyme comprises the following steps:
and 2, adding 125 mu L of the uniformly mixed compound M1-Fe and 8mg/mL PLGA-PEGTHF solution into THF, wherein the final volume is 4mL, adding 10mL of ultrapure water of a filter membrane under the ultrasonic condition, and carrying out ultrasonic treatment for 8min to obtain the nano enzyme.
Example 3
and 2, adding 125 mu L of the uniformly mixed compound M1-Fe and 8mg/mL PLGA-PEGTHF solution into THF, wherein the final volume is 4mL, adding 10mL of ultrapure water of a filter membrane under the ultrasonic condition, and carrying out ultrasonic treatment for 6min to obtain the nano enzyme.
Example 4
and 2, adding 93.75 mu L of the uniformly mixed compound M1-Fe and 8mg/mL PLGA-PEGTHF solution into THF, wherein the final volume is 4mL, adding 10mL of ultrapure water of a filter membrane under the ultrasonic condition, and performing ultrasonic treatment for 7min to obtain the nano enzyme.
Example 5
The concentration adopted in the M1-FeNPs measurement is 10-1μ g/mL, 4 μ L of the solution was dropped onto the silicon wafer, left at-20 ℃ for 1h, followed by freeze-drying for 2h, and the dried sample was used for SEM measurement. The results are shown in FIG. 1, and the particle size was measured to be about 90 nm.
Example 6
M1-FeNPs were diluted in 30. mu.g/mL aqueous solution, UV was measured to give an absorption maximum of 655nm, the measurement is shown in FIG. 2, blue-shifted by 15nm compared to M1 NPs.
Example 7
Performance testing of inorganic ion-mediated organic compound nanoenzyme production OH:
diluted with a buffer solution of pH 6.5, and the aminophenyl fluorescein solution was mixed with each component (H)2O2、M1NPs+H2O2、M1-FeNPs、M1-FeNPs+H2O2) Placing in a 37 deg.C incubator with final aminophenyl fluorescein concentration of 1mM, M1NPs/M1-FeNPs concentration of 50 μ g/mL, and H2O2The concentration is 10mM, wherein M1-FeNPs + H2O2The light irradiation group is at 660nm1W/cm2The fluorescence intensity was measured every 1min under irradiation. The measurement results are shown in FIG. 3, M1-FeNPs + H2O2Can generate a large amount of OH, and the generation of OH can be promoted by illumination.
Example 8
Testing the photo-thermal performance of the inorganic ion mediated organic compound nanoenzyme:
preparing nano enzyme (0-10 mug/mL) with different concentrations. 200 μ L of the mixture was put in a 0.5mL centrifuge tube and the laser intensity was 1W/cm2And irradiating the solution for 8min by using 660nm laser to obtain photothermal curves of the nanoenzymes with different concentrations. The measurement results are shown in FIG. 4.
Preparing nanoenzyme (M1-FeNPs) and organic compound M1NPs without inorganic ions, wherein the concentration of the NPs is 10 mug/mL, and 200 mug of the NPs are put into a 0.5mL centrifuge tube, and the laser intensity is 1W/cm respectively2Laser irradiation at 660nm for 8min, temperature was monitored and recorded. The laser was then turned off and the temperature of the solution was monitored until the original value was restored. The results are shown in FIG. 5, where M1-FeNPs could be raised to 55.2 ℃ and M1NPs to 52.2 ℃.
Preparing the nano enzyme with the concentration of 10 mu g/mL, putting 200 mu L into a centrifugal tube with the concentration of 0.5mL, and using laser with the intensity of 1W/cm2Laser irradiation at 660nm for 8min, temperature was monitored and recorded. The laser was then turned off and the temperature of the solution was monitored until the original value was restored. Repeat 5 times. The measurement results are shown in FIG. 6.
Example 9
Inorganic ion-mediated, organically combined nanoenzyme was tested for bactericidal effect against staphylococcus aureus (buffer solution used pH 6.5):
in a clean bench, 2mL of bacterial liquid is absorbed by staphylococcus aureus cultured in NB liquid medium for about 10 hours, centrifugation (7100rpm,2min) precipitation is carried out, the precipitated staphylococcus aureus is washed by 1 х PBS and then is centrifugally precipitated, the supernatant is discarded, the centrifugation is carried out twice, the supernatant is discarded, the bacterial liquid is resuspended in PBS, and OD is adjusted600Is 1.0. To a 1.5mL centrifuge tube, 100. mu.L (OD) was added6001) and a quantity of M1-FeNPs (final concentrations 2.5 μ g/mL, 5 μ g/mL, 7.5 μ g/mL, 10 μ g/mL, respectively), the volume was made up to 500 μ L with sterile 1 × PBS and incubated for 20min at 37 ℃ in the dark, two groups at each concentration, non-illuminated and illuminated, respectively, the blank group being not dosed. After the incubation, the light group was set at 660nm (1W/cm)2) Under the laser of (2), irradiation is carried out for 8 min. Dilution 1X 10 per group4After doubling, 100 mu L of bacterial liquid is sucked and evenly coated on a 90mmNB solid culture medium, the culture is carried out for 12h at 37 ℃, and the colony forming units are counted. (the test results are shown in FIG. 7, the survival rate of M1-FeNPs light-irradiated bacteria is 7.1% at 10. mu.g/mL under the condition of pH 6.5, and the sterilizing effect is best)
Example 10
Inorganic ion-mediated nanoenzyme bactericidal effect test on staphylococcus aureus (buffer solution used pH 7.4):
in a clean bench, 2mL of bacterial liquid is absorbed by staphylococcus aureus cultured in NB liquid medium for about 10 hours, centrifugation (7100rpm,2min) precipitation is carried out, the precipitated staphylococcus aureus is washed by 1 х PBS and then is centrifugally precipitated, the supernatant is discarded, the centrifugation is carried out twice, the supernatant is discarded, the bacterial liquid is resuspended in PBS, and OD is adjusted600Is 1.0. To a 1.5mL centrifuge tube, 100. mu.L (OD) was added6001) and a quantity of M1-FeNPs (final concentrations 2.5 μ g/mL, 5 μ g/mL, 7.5 μ g/mL, 10 μ g/mL, respectively), the volume was made up to 500 μ L with sterile 1 x PBS and incubated for 20min at 37 ℃ in the dark, two groups at each concentration, non-illuminated and illuminated, respectively, blank group withoutAdding medicine. After incubation, non-illuminated groups were diluted 1X 104After doubling, 100 mu L of bacterial liquid is sucked and evenly coated on a 90mmNB solid culture medium, the culture is carried out for 12h at 37 ℃, and the colony forming units are counted. The light set is 660nm (1W/cm)2) Under the laser of (2), irradiation is carried out for 8 min. (the test results are shown in FIG. 7, and the survival rate of M1-FeNPs light-irradiated bacteria is 30.8% at 10. mu.g/mL).
Claims (7)
1. An inorganic ion mediated organic compound nanoenzyme, wherein the nanoenzyme is prepared by the following steps:
step 1, dissolving a compound M1 and inorganic ions in tetrahydrofuran, stirring at room temperature, and uniformly mixing to obtain a compound M1-Fe;
step 2, dissolving a compound M1-Fe and polylactic acid-glycolic acid copolymer-polyethylene glycol in tetrahydrofuran, adding the mixture into ultrapure water of a filter membrane under an ultrasonic condition, and performing ultrasonic treatment for 5-8 min to obtain the nano enzyme;
the inorganic ion is Fe2+Said compound M1 is:
2. the inorganic ion mediated organic compound nanoenzyme according to claim 1, wherein the molar ratio of the compound M1 to the inorganic ions in the step 1 is 1: 1-3, and the stirring time at room temperature is 2-4 h.
3. The inorganic ion-mediated organic compound nanoenzyme of claim 1, wherein the mass ratio of M1-Fe to polylactic acid-glycolic acid copolymer-polyethylene glycol in the step 2 is 1: 4-8.
4. The method for preparing the inorganic ion mediated organic compound nanoenzyme according to claim 1, comprising the steps of:
step 1, dissolving a compound M1 and inorganic ions in tetrahydrofuran, stirring at room temperature, and uniformly mixing to obtain a compound M1-Fe;
step 2, dissolving a compound M1-Fe and polylactic acid-glycolic acid copolymer-polyethylene glycol in tetrahydrofuran, adding the mixture into ultrapure water of a filter membrane under an ultrasonic condition, and performing ultrasonic treatment for 5-8 min to obtain the nano enzyme;
the inorganic ion is Fe2+Said compound M1 is:
5. the method for preparing the inorganic ion mediated organic compound nanoenzyme according to claim 4, wherein the molar ratio of the compound M1 to the inorganic ions in the step 1 is 1: 1-3, and the stirring time at room temperature is 2-4 h.
6. The method for preparing the inorganic ion mediated organic compound nanoenzyme according to claim 4, wherein the mass ratio of M1-Fe to the polylactic acid-glycolic acid copolymer-polyethylene glycol in the step 2 is 1: 4-8.
7. The use of an inorganic ion-mediated organic compound nanoenzyme according to claim 1, in the preparation of a medicament for the treatment of a bacterial infection.
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