Selenium-containing nano HOF, antioxidant nanozyme, preparation method and application thereof

Abstract

A multi-enzyme cascade antioxidant nano HOF and preparation method and application thereof are provided. The present invention discloses a nano HOF based on selenium-containing ligand for the first time, the nano HOF does not contain metal and has good biocompatibility, which is a universal loading platform, and has high porosity, acid and alkali resistance and thermal stability, and is size-tunable, it can coat various types of enzymes at the same time, stabilize the conformation of the enzyme through the confinement effect, the high porosity can not only provide enough space for the enzyme, but also facilitate the transport of substances and the play of catalytic properties of the enzyme, 85-90% of the activity of the coated enzyme can be maintained, ROS can be effectively scavenged through the cascade catalysis between various enzymes, which provides a new idea for the construction of bio-friendly antioxidants.

Claims

1. A nano hydrogen-bonded organic framework (HOF), wherein a morphology of the nano HOF is a rod-like structure, with a positive charge on a surface of the rod-like structure, and a size of the nano HOF is 10 m-300 nm; the nano HOF is formed by reacting an amidine-containing compound and carboxyl groups of a selenium-containing ligand, wherein a structural formula of the amidine-containing compound is Formula (I), and a structural formula of the selenium-containing ligand is Formula (II), ##STR00004## wherein R of the Formula (II) is one of H, F, Br, OH, and CH.sub.3.

2. A method for preparing an antioxidant nanozyme comprising a step of adding the nano HOF according to claim 1 to an enzyme, wherein the nano HOF is a loading platform, and the enzyme is an antioxidant enzyme.

3. A multi-enzyme cascade antioxidant nanozyme comprising the nano HOF according to claim 1 and an antioxidant enzyme.

4. A method for reducing a concentration of reactive oxygen species (ROS) in cells comprising a step of adding the multi-enzyme cascade antioxidant nanozyme according to claim 3 to a cell.

5. The multi-enzyme cascade antioxidant nanozyme according to claim 3, wherein the enzyme is one or two of glutathione peroxidase, catalase, or superoxide dismutase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) To explain the embodiments of the present invention or the technical solutions in the prior art more clearly, a brief introduction will be made to the accompanying drawings used in the embodiments. It is obvious that the drawings in the description below are only some embodiments of the present invention, and those ordinarily skilled in the art can obtain other drawings according to these drawings without creative work.

(2) FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of amidine-containing compound prepared by embodiment 1 of the present invention;

(3) FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of selenium-containing ligand prepared by embodiment 2 of the present invention;

(4) FIG. 3 is a scanning electron microscope image of nano HOF based on selenium-containing ligand prepared by embodiment 3 of the present invention;

(5) FIG. 4 is an apparent zeta potential diagram of nano HOF based on selenium-containing ligand prepared by embodiment 3 of the present invention;

(6) FIG. 5 is a result of glutathione peroxidase activity test of nano HOF based on selenium-containing ligand in embodiment 4 of the present invention;

(7) FIGS. 6A-6D are scanning electron microscope images of four kinds of selenium-containing nano HOF aqueous solution in the experimental test 2 of the present invention, wherein FIG. 6A is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 0.1 mg/mL of amidine-containing compound solution;

(8) FIG. 6B is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 0.2 mg/mL of amidine-containing compound solution; FIG. 6C is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 0.5 mg/mL of amidine-containing compound solution; FIG. 6D is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 1 mg/mL of amidine-containing compound solution;

(9) FIG. 7 is a scanning electron microscope image of a multi-enzyme cascade antioxidant nanozyme based on the selenium-containing ligand nano HOF prepared by embodiment 6 of the present invention;

(10) FIG. 8 is a result of a superoxide dismutase activity test in experimental test 3 of the present invention;

(11) FIG. 9 is a result of a catalase activity test in experimental test 4 of the present invention;

(12) FIG. 10 is a result of a cytotoxicity test in experimental test 5 of the present invention;

(13) FIG. 11 is a result of ROS scavenging capacity in vitro in experimental test 6 of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(14) The technical solution of the present invention will be further elaborated hereafter in conjunction with accompanying drawings and embodiments.

(15) In order to make the objectives, the technical solutions, and the advantages of the application more clear, thorough and complete, the technical scheme of the present invention is described clearly and completely through the attached drawings and embodiments. The following detailed descriptions are descriptions of the embodiments, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those commonly understood by the general technical personnel in the field to which the application belongs.

(16) The instruments and reagents used in the embodiments are all commercially available.

Embodiment 1

(17) The preparation for amidine-containing compound as shown in Formula (I), the specific steps are as follows:

(18) S1.1, 10.17 g of copper cyanide and 13.63 g of tetrakis (4-bromophenyl) methane were added into 80 mL of N, N-Dimethylformamide (DMF) and reacted at 140 C. for 48 h under a nitrogen atmosphere.

(19) S1.2, after the reaction, DMF was spin-dried, and extracted with dichloromethane, and then washed with 1 mM of sodium ethylenediaminetetraacetate aqueous solution and saturated salt water, respectively. The organic layer was dried by anhydrous magnesium sulfate, and after filtration and evaporation, the obtained crude product was purified by column chromatography to obtain a white solid.

(20) S1.3, the 0.5 g of white solid obtained by step S1.2 was dissolved in 10 mL of dry tetrahydrofuran (THF), cooled to 78 C. with liquid nitrogen under the protection of nitrogen, and then THE solution of the lithium bis(trimethylsilyl)amide (LiHMDS) (7.1 mL, 1.0 M) was added dropwise, and the precipitate was obtained immediately. The mixture was placed at room temperature, stirred overnight, and the precipitate was slowly dissolved to obtain an orange solution.

(21) After the above solution was cooled to 0 C., 12 mL of liquid (2 mL of acetyl chloride and 10 mL of ethanol) was added to form a grayish-white precipitate. Amidine-containing compound can be obtained as shown in Formula (I) after filtered, fully washed with anhydrous ethanol, and vacuum dried, the nuclear magnetic resonance hydrogen spectrum of amidine-containing compound is shown in FIG. 1.

(22) ##STR00002##

Embodiment 2

(23) The preparation for selenium-containing ligand as shown in Formula (II), the specific steps are as follows:

(24) S2.1, 2.5 g of p-aminobenzoic acid and 1 mL of concentrated hydrochloric acid were added to a round-bottom flask containing 3 mL of water, and mixed well to obtain a hydrochloric acid solution of p-aminobenzoic acid. The 0.6 g of sodium nitrite was dissolved in 2 mL of water, and then added dropwise to the prepared hydrochloric acid solution of p-aminobenzoic acid at the rate of 5 s/drop, after dropping, the reaction was continued for 5 min to obtain a clear pale yellow solution.

(25) S2.2, 0.5 g of selenium powder and 1.7 g of sodium borohydride were added to the round-bottom flask, and 5 mL of water was added under the protection of nitrogen, after no bubbles were generated, the clear yellow solution obtained by step S2.1 was slowly added dropwise, the reaction was continued for 4 h after dropped. After the reaction, concentrated hydrochloric acid was added dropwise to the solution until the yellow solid no longer precipitate, filtered, and the precipitate was taken, the selenium-containing ligand can be obtained as shown in Formula (II) after washed with ultrapure water, dried, and purified by column chromatography, the structural formula is

(26) ##STR00003##
the nuclear magnetic resonance hydrogen spectrum of selenium-containing ligand is shown in FIG. 2.

Embodiment 3

(27) The amidine-containing compound prepared by embodiment 1 and the selenium-containing ligand prepared by embodiment 2 were used to prepare the selenium-containing ligand nano HOF, the specific steps are as follows:

(28) S3.1, 10 mg of the amidine-containing compound shown in Formula (I) prepared by embodiment 1 was added to 10 mL of deionized water and stirred for 5 min to obtain an amidine-containing compound solution.

(29) S3.2, 1.5 mg of selenium-containing ligand shown in Formula (II) prepared by embodiment 2 was dissolved in 2 mL of deionized water, and then the pH of the solution was adjusted to 10 with 2M of sodium hydroxide solution to obtain selenium-containing ligand solution.

(30) S3.3, under the condition of 20 C. and stirred, the selenium-containing ligand solution prepared by step S3.2 was added dropwise to the amidine-containing compound solution prepared by step S3.1 at a rate of 10 s/drop, after stirring for 2 h, it was centrifuged at 5000 RPM for 10 min at 4 C., and washed with ultrapure water for 3 times, the precipitate was redispersed into ultrapure water to obtain a selenium-containing nano HOF aqueous solution, the scanning electron microscope image is shown in FIG. 3, and the apparent zeta potential was tested by a Malvern nano-particle size analyzer, the results are shown in FIG. 4.

(31) It can be seen from FIG. 3 and FIG. 4 that the synthesized nano HOF has a rod-like structure of about 300 nm, and its apparent zeta potential is about +10 mV.

Experimental Test 1

(32) The glutathione peroxidase (GPx) activity test is carried out for nano HOF based on the selenium-containing ligand prepared in embodiment 3, the specific steps are as follows:

(33) S4.1, the GPx activity was measured by coupling reductase method, the mixture solution containing 50 mM of phosphate buffer (pH7.4, 1.0 mM of ethylenediaminetetraacetic acid (EDTA)), 1.0 U.Math.mL.sup.1 of glutathione reductase (GR), 1.0 mM of glutathione (GSH) and different concentrations of selenium-containing nano HOF aqueous solution (concentrations were 0.259 g/mL, 0.518 g/mL, 0.777 g/mL, 1.036 g/mL, 1.295 g/mL, and 2.590 g/mL, respectively) was incubated at 37 C. for 3 min, and then 0.25 mM of nicotinamide adenine dinucleotide phosphate (NADPH) solution was added, and continued to incubate for 1 min. Finally, 0.5 mM of H.sub.2O.sub.2 was added to start the reaction.

(34) S4.2, the absorption value of NADPH at 340 nm (F=6220M.sup.1 cm.sup.1, pH=7.4) was measured by using an ultraviolet/visible spectrophotometer, and the GPx activity was determined according to the change of the absorption value, the absorption value is shown in FIG. 5.

(35) It can be seen from FIG. 5 that with the increase of the concentration of selenium-containing nano HOF, the activity of GPx is also gradually enhanced, and the selenium-containing nano HOF exhibits a concentration-dependent characteristic of the enzyme, indicating that the selenium-containing nano HOF has good glutathione peroxidase activity.

Experimental Test 2

(36) The size tunability of nano HOF based on the selenium-containing ligand prepared in embodiment 3 was investigated, the specific steps are as follows:

(37) S5.1, 1 mg, 2 mg, 5 mg, and 10 mg of amidine-containing compounds shown in Formula (I) prepared by embodiment 1 were added to 10 mL of deionized water respectively, and stirred for 5 min, the amidine-containing compound solutions with four concentrations of 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, and 1 mg/mL were obtained.

(38) S5.2, the 6 mg of selenium-containing ligand shown in Formula (II) prepared by embodiment 2 was dissolved in 8 mL of deionized water, and then the solution pH was adjusted to 10 with 2M of sodium hydroxide solution to obtain a selenium-containing ligand solution.

(39) S5.3, under the condition of 20 C. and stirring, 2 mL of selenium-containing ligand solution prepared by step S5.2 was added dropwise to the four concentrations of amidine compound solution prepared by step S5.1 at a rate of 10 s/drop, after stirring for 2 h, it was centrifuged at 5000 RPM for 10 min at 4 C., and washed with ultrapure water for 3 times, the precipitate was redispersed into ultrapure water to obtain four selenium-containing nano HOF aqueous solutions, the scanning electron microscope image of four kinds of selenium-containing nano HOF aqueous solution are shown in FIGS. 6A-6D, wherein FIG. 6A is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 0.1 mg/mL of amidine-containing compound solution; FIG. 6B is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 0.2 mg/mL of amidine-containing compound solution; FIG. 6C is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 0.5 mg/mL of amidine-containing compound solution; FIG. 6D is a scanning electron microscope image of selenium-containing nano HOF aqueous solution prepared by 1 mg/mL of amidine-containing compound solution.

(40) It can be seen from FIGS. 6A-6D that the size of selenium-containing nano HOF prepared by 0.1 mg/mL of amidine-containing compound solution is about 10 m, the size of selenium-containing nano HOF prepared by 0.2 mg/mL amidine-containing compound solution is about 6 m, the size of selenium-containing nano HOF prepared by 0.5 mg/mL amidine-containing compound solution is about 3 m, and the size of selenium-containing nano HOF prepared by 1 mg/mL amidine-containing compound solution is about 300 nm. It is shown that applying the nano HOF based on selenium-containing ligand prepared by the method provided by the present invention is size-tunable.

Embodiment 4

(41) A multi-enzyme cascade loaded superoxide dismutase nanozyme based on selenium-containing nano HOF was prepared, the specific steps are as follows:

(42) S5.1, 10 mg of the amidine-containing compound shown in Formula (I) prepared by embodiment 1 was added to 10 mL of deionized water and stirred for 5 min to obtain an amidine-containing compound solution. A superoxide dismutase solution was prepared by dissolving 0.1 mg of superoxide dismutase in 1 mL of ultrapure water.

(43) S5.2, 1.5 mg of selenium-containing ligand shown in Formula (II) prepared by embodiment 2 was dissolved in 2 mL of deionized water, and then the pH of the solution was adjusted to 10 with 2M of sodium hydroxide solution to obtain selenium-containing ligand solution.

(44) S5.3, under the condition of 20 C. and stirred, the enzyme solution was added dropwise to the solution containing amidine compound prepared by step S5.1 at the rate of is/drop, after stirring for 10 min, the selenium-containing ligand solution was added dropwise at the rate of 10 s/drop, after stirring for 2 h, it was centrifuged at 5000 RPM for 10 min at 4 C., and washed with ultrapure water for 3 times, the precipitate was redispersed into ultrapure water to obtain a multi-enzyme cascade antioxidant nanozyme aqueous solution, which was recorded as SeHOF@SOD.

Embodiment 5

(45) A multi-enzyme cascade nanozyme loaded catalase based on selenium-containing nano HOF was prepared, the method was exactly the same as that of Embodiment 4, only the added enzyme was catalase, and the obtained multi-enzyme cascade antioxidant nanozyme aqueous solution was recorded as SeHOF@CAT.

Embodiment 6

(46) A multi-enzyme cascade nanozyme loaded superoxide dismutase and catalase based on selenium-containing nano HOF was prepared, the method was the same as that of Embodiment 4, only the added enzymes were superoxide dismutase and catalase, the additional amount of superoxide dismutase and catalase was 0.1 mg, and the obtained multi-enzyme cascade antioxidant nanozyme aqueous solution was recorded as SeHOF@CAT@SOD.

(47) The scanning electron microscope image is shown in FIG. 7, after the selenium-containing nano HOF is loaded with superoxide dismutase and catalase, the rod-like structure is still maintained, and its size is about 300 nm, which proves the successful construction of multi-enzyme cascade antioxidant nanozymes.

Experimental Test 3

(48) The superoxide dismutase activity test of the multi-enzyme cascade antioxidant nanozyme prepared in embodiment 4 was tested, the steps are as follows:

(49) SOD activity was quantified by using a standard xanthine/xanthine oxidase (XOD) assay system developed by McCord and Fridovich. The xanthine/XOD system produced superoxide radical anion at pH=7.0 (phosphate buffer, 25 C.). Appropriate amount of antioxidant nanozyme (0.248 g/mL, 0.645 g/mL, 1.24 g/mL, 2.48 g/mL, 4.96 g/mL, and 12.4 g/mL) was added to an aqueous solution containing XOD (final concentration is 0.025 U.Math.mL.sup.1), 100 mM of nitro blue tetrazolium (NBT), 300 mM of xanthine, 0.1 mM of EDTA and 50 mM of phosphate buffer (final volume is 500 L) and the oxidation of the water-soluble tetrazolium salt WST-8 was monitored at 450 nm using a UV/Vis spectrophotometer. SOD activity was evaluated by using the enzyme concentration at which the oxidation rate of WST-8 was inhibited by 50% (IC.sub.50).

(50) As shown in FIG. 8, with the increase in the concentration of antioxidant nanozymes, the oxidation inhibition rate of WST-8 was higher and higher, indicating that the scavenging effect of superoxide radicals was better and better, which proved that the antioxidant nanozymes had good superoxide dismutase activity, and the IC.sub.50 value was 0.53 g/mL.

Experimental Test 4

(51) The catalase activity of the multi-enzyme cascade antioxidant nanozyme prepared by embodiment 5 and embodiment 6 was tested, the method is as follows:

(52) The catalase (CAT) mimic activity of antioxidant nanozymes was determined by monitoring the change of O.sub.2 concentration by using a portable dissolved oxygen meter (JPB-607A, Leici China), all reactions were performed in 0.1 M of phosphate buffer (pH 7.4) at 25 C., and the results are shown in FIG. 9.

(53) It can be seen from FIG. 9 that with the passage of time, the oxygen content in the solution is getting higher and higher, indicating that the antioxidant nanozyme can effectively decompose hydrogen peroxide into oxygen, which proves that the antioxidant nanozyme has good catalase activity.

Experimental Test 5

(54) The cytotoxicity of the multi-enzyme cascade antioxidant nanozymes prepared by embodiments 4 to 6 was tested, the method is as follows:

(55) Cell Counting Kit-8 (CCK-8) was used to quantitatively detect the effect of nanozymes on cell viability. PC12 cells were seeded in 96-well plates at a density of 5000 cells per well (total volume of 180 mL per well) for 24 h. Then, different concentrations of nanozymes (6.25 g/mL, 12.5 g/mL, 25 g/mL, 50 g/mL, and 100 g/mL) were added to the cell culture medium. The cells were incubated with multi-enzyme cascade nanozymes for 24 h.

(56) In order to determine the toxicity, 20 L of CCK-8 solution was added to each well and the plate was incubated in a CO.sub.2 incubator for 30 min. Then the solution is transferred from the 96-well plate to a new 96-well plate. Then the 96-well plate was rotated at room temperature for 30 s in the dark. The absorption value was measured at 450 nm by using a Bio-Rad 680 microplate reader. Cell viability was estimated according to the following equation: cell viability (%)=(OD treatment/OD control) 100%. The OD control was obtained in the absence of multi-enzyme cascade nanozymes, and the OD treatment was obtained in the presence of multi-enzyme cascade nanozymes. The results are shown in FIG. 10.

(57) It can be seen from FIG. 10 that the cell survival rate is above 90%, indicating that the multi-enzyme cascade nanozyme has good biocompatibility and is conducive to its biological application.

Experimental Test 6

(58) The ROS scavenging capacity in vitro of multi-enzyme cascade antioxidant nanozymes prepared from embodiments 4 to 6 was tested, the method is as follows:

(59) The production of ROS was monitored by using 2,7-dichlorofluorescein diacetate (DCFH-DA), which reacts with intracellular free radicals and produces a fluorescent substance 2,7-dichlorofluorescein (DCF). The intensity of DCF fluorescence is related to the level of ROS in cells. In order to carry out the experiment, 3T3 cells were inoculated in 6-well plates and incubated for 24 h. The cell culture medium was removed, and then the adherent cell PC12 was incubated with different nanozymes at 37 C. for 4 h. Before using, the adherent cell PC12 cells were washed three times with phosphate buffer saline (PBS) (pH 7.4) to remove the excess nanozymes. Then the cells were incubated with Rosup (50 g.Math.mL.sup.1) at 37 C. for 1 h.

(60) After that, DCFH-DA solution was added to PC12 cells and the mixture was incubated at 37 C. for 1 h. Cells were collected and carried out the data acquisition and analysis on a flow cytometry. The excitation wavelength is 488 nm, and the signal is collected at channel 2 (500-560 nm). The results are shown in FIG. 11.

(61) It can be seen from FIG. 11 that antioxidant nanozymes can effectively reduce the content of ROS in cells, and nano HOF loaded with superoxide dismutase and catalase showed the highest scavenging efficiency of ROS, with a scavenging rate of 88%, and showed the advantage of cascade antioxidant.

(62) The present invention discloses a nano HOF based on selenium-containing ligand for the first time, the nano HOF does not contain metal and has good biocompatibility, which is a universal loading platform, and has high porosity, acid and alkali resistance and thermal stability, and is size-tunable, it can coat various types of enzymes at the same time, stabilize the conformation of the enzyme through the confinement effect, the high porosity can not only provide enough space for the enzyme, but also facilitate the transport of substances and the play of catalytic properties of the enzyme, 85-90% of the activity of the coated enzyme can be maintained; ROS can be effectively scavenged through the cascade catalysis between various enzymes, which provides a new idea for the construction of bio-friendly antioxidants and a new paradigm for the treatment of related diseases induced by continuous oxidative stress.

(63) Finally, it should be noted that the above embodiments are merely used for describing the technical solutions of the present invention, rather than limiting the same. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention may still be modified or equivalently replaced. However, these modifications or substitutions should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.