CN114890385B - Efficient antioxidant two-dimensional hydrogen germanium alkene nano-sheet and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a high-efficiency antioxidant two-dimensional hydrogen germanium alkene nano-sheet, and a preparation method and application thereof. The two-dimensional germanium nano-sheet has an ultrathin layered structure, and the surface of the two-dimensional germanium nano-sheet is covalently modified with H atoms.
Description
Technical Field
The invention relates to an ultrathin germanium nanosheet (Germanene) with excellent oxidation resistance, uniform size and high specific surface area, and hydrogen (H) atoms connected to the surface, a preparation method thereof and related application of the ultrathin germanium nanosheet (Germanene) serving as an efficient and low-toxicity biomedical two-dimensional nanomaterial, and belongs to the technical field of two-dimensional nanomaterials.
Background
Reactive Oxygen Species (ROS), including superoxide anions (O) 2 ·- ) Hydrogen peroxide (H) 2 O 2 ) Singlet oxygen 1 O 2 ) And hydroxyl radicals (. OH) play a vital role in regulating various physiological functions of organisms, and are involved in mechanisms necessary for the growth, health or aging of organisms, often playing an important role as signal molecules. However, ROS also have some potentially toxic effects, including oxidation and damage to DNA, RNA, proteins, and membranes, which requires intracellular antioxidant systems to maintain ROS at substantially non-toxic levels, any deviation from this balance may lead to disease onset (Trends)The inoplant Science,2016,22 (1), 11-19). Acute Kidney Injury (AKI), a disease associated with oxidative stress, results in clinically high mortality rates annually (adv.funct.mater.2019, 29,1904833). While the importance of inhibiting oxidative stress has been recognized for decades, antioxidant therapy has been greatly delayed due to the lack of safe and effective drug candidates. For this reason, a large number of researchers have designed various functional Nano-drugs for the antioxidant treatment of AKI, such as Black Phosphorus (BP) (Nano Lett.2020,20, 1447-1454), cu 5.4 O nanoparticles (Nature Communications,2020,11 (1)), and the like.
Disclosure of Invention
In view of the above problems, the present invention aims to provide an ultrathin germanium nanoplatelet with excellent oxidation resistance, and a preparation method and application thereof.
In one aspect, the present invention provides a two-dimensional germanium nanoplatelet having an ultra-thin layered structure with surface covalently modified H atoms (i.e., germanene-H, two-dimensional GeH nanoplatelets).
In the present disclosure, the two-dimensional GeH nanoplatelets having an ultra-thin layered structure have excellent oxidation resistance and can scavenge a range of representative Reactive Oxygen Species (ROS), including hydrogen peroxide (H) 2 O 2 ) Superoxide anion (O) 2 ·- ) And hydroxyl radical (·oh) and the like, can be well applied to the treatment of AKI diseases associated with ROS.
Preferably, the transverse dimension of the two-dimensional germanium nano-sheet is 200-300nm, and the thickness is 0.5-2 nm.
Preferably, the two-dimensional germanium nanoplatelets have a PH-responsive degradation behavior that can be stable in acidic (PH < 7, inflammatory microenvironment) solutions for a longer period of time, with a linear relationship between the two as PH decreases and rapid degradation at neutral (ph=7.4, normal tissue microenvironment) times. The characteristic of selective degradation can realize the aim of inflammation targeted therapy, and has better biological safety.
Preferably, the two-dimensional germanium nano-sheet further comprises polyvinylpyrrolidone modified on the surface of the two-dimensional germanium nano-sheet, and the mass ratio of the two-dimensional germanium nano-sheet to the polyvinylpyrrolidone is 1: (10-20). The in vivo biological application of the material can be better realized through the surface modification of PVP.
On the other hand, the invention also provides a preparation method of the two-dimensional germanium nano-sheet, which comprises the following steps:
(1) The precursor CaGe with lamellar microstructure is prepared 2 Carrying out rapid heating treatment and quenching treatment, and then immersing in concentrated hydrochloric acid solution for magnetic stirring to obtain a mixed solution;
(2) And centrifuging the obtained mixed solution, removing supernatant, and cleaning precipitate by using anhydrous acetonitrile or acidic deionized water solution to obtain the two-dimensional germanium nanosheets.
Preferably, the temperature of the rapid heat treatment is 360-400 ℃.
Preferably, the temperature of the quenching treatment is-196 ℃.
Preferably, the concentration of the concentrated hydrochloric acid solution is 10 to 13moL/L, preferably 12moL/L.
Preferably, the temperature of the magnetic stirring is-40 ℃, the rotating speed is 500-600 rpm, and the time is 6-8 days.
Preferably, the rotation speed of the centrifugal treatment is 18000-20000 revolutions per minute, and the time is 10-30 minutes. Preferably, the rotational speed of the centrifugal treatment is 19000 revolutions per minute, and the time is 15-30min.
In yet another aspect, the invention also provides an application of the two-dimensional germanium nanoplatelets in preparing AKI disease treatment materials related to ROS.
The beneficial effects are that:
the preparation method disclosed by the invention is simple in preparation process, high in feasibility, low in cost and high in yield, and the obtained nano material is controllable in size and excellent in performance, and can be used as an excellent antioxidant for ROS (reactive oxygen species) removal related biological application.
In the nanomaterial system, the H atoms connected with the surface of the GeH nanosheets can effectively remove Reactive Oxygen Species (ROS) to achieve the purpose of relieving Acute Kidney Injury (AKI).
Drawings
FIG. 1 is a flow chart of a two-dimensional GeH nanoplatelet prepared in example 1;
FIG. 2 is a schematic diagram of a precursor CaGe for two-dimensional GeH nanoplatelet synthesis in example 1 2 As can be seen from SEM images of (a), it has a distinct layered structure;
FIG. 3 is a TEM image of a two-dimensional GeH nanoplatelet of example 1, which has uniform size and good dispersibility (scale, 200 nm);
FIG. 4 is an Atomic Force Microscope (AFM) image of two-dimensional GeH nanoplatelets of example 1, demonstrating that the nanoplatelets have an ultra-thin structure, with a thickness of about 1 nm;
FIG. 5 is an infrared absorption spectrum of a two-dimensional GeH nanoplatelet of example 1, demonstrating the presence of covalently attached hydrogen atoms on the nanoplatelet surface;
FIG. 6 is a Raman spectrum of a two-dimensional GeH nanoplatelet of example 1, demonstrating the presence of covalently attached hydrogen atoms on the nanoplatelet surface;
FIG. 7 shows the formation of hydroxyl radicals (. OH) and superoxide anions (O) in the presence of water and oxygen in the two-dimensional GeH nanoplatelets of example 1 2 ·- ) The Electron Spin Resonance (ESR) test results before and after reaction prove that the two-dimensional GeH nanosheets can effectively remove ROS and are possibly applied to treating inflammatory diseases related to ROS;
FIG. 8 is a cytotoxicity test result of the two-dimensional GeH nanoplatelets in example 1, which proves that the material has no toxicity to human embryonic kidney source 293 (HEK 293) cells and good biological application potential;
FIG. 9 is a two-dimensional GeH nanosheet pair and H in example 1 2 O 2 Protection results of co-incubated HEK293 cells.
Detailed Description
The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof.
In the present disclosure, the two-dimensional GeH nanoplatelets with hydrogen atoms modified on the surface can consume ROS by the hydrogen atoms connected to the surface, and the reduction characteristic of Ge can also realize the reaction with ROS, thereby achieving the purpose of scavenging. Meanwhile, the two-dimensional GeH nanosheets are used as nano drug carrier materials, the high specific surface area of the two-dimensional GeH nanosheets greatly improves the ROS scavenging efficiency, and the high-efficiency and low-toxicity treatment effect is achieved.
In the invention, the preparation method of the two-dimensional GeH nanosheets has simple and feasible synthesis process and controllable and accurate reaction conditions. Fig. 1 is a flowchart of preparing a two-dimensional GeH nanosheet according to an embodiment of the present invention. The method of preparing the two-dimensional GeH nanoplatelets having hydrogen atoms modified on the surface is exemplarily described as follows.
The Ca block and the Ge powder are mixed according to the stoichiometric ratio of 1:2, after weighing, uniformly mixing and placing the mixture into a quartz tube coated with a carbon film. Then, while vacuumizing, sealing the tube by using oxyhydrogen flame. Putting a quartz tube filled with materials into a muffle furnace, heating to 900-1200 ℃ within 1-2 h, annealing for 16-20 h under the condition, and naturally cooling to room temperature to obtain a precursor CaGe 2 。
The precursor CaGe 2 Grinding into powder to obtain CaGe powder 2 。
Powdered CaGe 2 Through a thermal expansion treatment (for example, heating to 380 ℃) and then immersing it in liquid N 2 (L-N 2 ) In, until L-N 2 Complete gasification (e.g., quenching to-196 ℃) gives a precursor CaGe with a layered microstructure 2 And (3) powder.
The precursor CaGe with lamellar microstructure is prepared 2 Immersing the powder in concentrated hydrochloric acid solution, magnetically stirring under low temperature, and adding HCl and CaGe 2 Reacting to generate CaCl 2 Thereby effectively etching away the Ca layer while leaving a separate Ge layer.
The mixed solution was subjected to high-speed centrifugation and the supernatant (containing the reaction by-product CaCl) was removed 2 Etc.), collecting the precipitate, and washing with anhydrous acetone or acidic deionized water solution for multiple times (for example, three times) to obtain the two-dimensional GeH nanometer sheet.
According to the invention, the ultrathin two-dimensional GeH nano-sheet with good stability, uniform size, safety and low toxicity is synthesized by using the simple and easy and environment-friendly method. The GeH nano-sheet has an ultrathin structure, the transverse dimension is 200-300nm, and the thickness is about 1 nm.
In the invention, the two-dimensional GeH nano-sheet can exist stably in weak acid microenvironment of inflammation, and is easy to degrade in neutral condition of normal tissues. The inflammation treatment is targeted, the biological safety is improved, and the possibility of systemic toxicity caused by systemic distribution is avoided. Meanwhile, due to the reducibility of Ge, the Ge can react with ROS, the oxidation stress level of an inflammation part is reduced, and the hydrogen atoms connected with the surface of the Ge further enhance the reactivity, so that the efficient inflammation treatment effect is achieved.
The present invention will be further illustrated by the following examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.
Example 1
Preparing a two-dimensional GeH nano sheet: 1.092g of Ca block and 3.919g of Ge powder are weighed respectively, mixed uniformly and added into a quartz tube coated with a carbon film. And sealing the pipe by utilizing oxyhydrogen flame under the condition of fully vacuumizing. The quartz tube after tube sealing operation is put into a muffle furnace, the temperature is raised to 1000 ℃ at the heating rate of 10 ℃/min, the quartz tube is annealed for 18 hours under the condition, and then the quartz tube is naturally cooled to room temperature, thus obtaining the blocky CaGe 2 . The obtained bulk CaGe 2 Further grinding into powder, weighing 1g of powdered CaGe 2 It is subjected to a thermal expansion treatment again (heating to 380 ℃) and then immersed in liquid N 2 (L-N 2 ) In, until L-N 2 Complete gasification (quenching to-196 ℃) gives the precursor CaGe with a lamellar microstructure 2 And (3) powder. The precursor CaGe with lamellar microstructure is prepared 2 The powder was immersed in 100ml of concentrated hydrochloric acid solution (12 moL/L) and magnetically stirred at 600rpm at-40℃for 24 hours. Then high-speed centrifuging (19000 rpm,20 min), collecting precipitate, and washing with anhydrous acetone or acidic deionized water solution for three timesTo obtain the product GeH nanosheets. Further, 1mg (dissolved in 5mL of ethanol) of GeH was mixed with 10mg (dissolved in 5mL of ethanol) of PVP, followed by stirring (500 rpm,6 hours) in a water bath at 50℃and finally, the precipitate was collected by centrifugation, whereby PVP-modified GeH (PVP-GeH) was obtained.
FIG. 2 is a schematic diagram of a precursor CaGe of a two-dimensional GeH nanosheet of example 1 2 Is shown visually by SEM pictures of CaGe 2 Has a layered microstructure, illustrating the precursor CaGe 2 Is a layered compound in which Ca layers and Ge layers are alternately arranged by a close interlayer force.
FIG. 3 shows that the two-dimensional GeH nanoplatelets synthesized in example 1 can weaken the bonding force between the inner layers of the precursor in FIG. 2 by reducing the concentration of the precursor by quenching and quenching treatment, and further the Ca layer can be effectively removed by concentrated hydrochloric acid treatment, so that the Ge layer with good dispersibility and uniform size can be reserved.
FIG. 4 is an Atomic Force Microscope (AFM) image of a two-dimensional GeH nanoplatelet of example 1, showing that it has a thickness of about 1nm, has an ultra-thin structure, and conforms to the characteristics of the nanoplatelets.
FIG. 5 is an infrared absorption spectrum of a two-dimensional GeH nanosheet in example 1, which demonstrates that covalent modification of hydrogen atoms on the nanosheet surface was successfully achieved, and a GeH nanosheet was synthesized.
Fig. 6 is a raman spectrum of the two-dimensional GeH nanoplatelets of example 1, which demonstrates that covalent modification of hydrogen atoms on the nanoplatelets surface was successfully achieved, and GeH nanoplatelets were synthesized.
FIG. 7 shows the formation of hydroxyl radicals (. OH) and superoxide anions (O) in the presence of water and oxygen in the two-dimensional GeH nanoplatelets of example 1 2 ·- ) The Electron Spin Resonance (ESR) test results before and after the reaction prove that the two-dimensional GeH nano-sheet can effectively remove the ROS.
The cytotoxicity test of the GeH nanoplatelets in this example 1 was evaluated using a classical CCK-8 kit. In CCK-8 experiments, cells were first incubated at 1X 10 4 Density of wells/wells was inoculated into 96-well plates and then incubated at 37℃with 5% CO 2 CO of humid air 2 Culturing in an incubator for 24h to allow the cells to adhere to the wall. Next, PVP-GeH (40. Mu.g/mL, 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL) was used at various concentrationsg/mL, where concentration is based on the mass of Ge) replaces the medium in the adherent cells and incubation is continued for an additional 24h. After the incubation was completed, the broth was removed and washed 3 times with fresh broth. Adding ten times of CCK-8 solution diluted by culture medium into each well, and placing at 37deg.C with 5% CO 2 CO of humid air 2 Incubate for another 4h in incubator. Finally, the absorbance (λ=450 nm) was measured on a microplate reader. Cytotoxicity index is expressed as the percentage of cell viability after sample treatment relative to that of untreated blank. Fig. 8 is a graph showing cytotoxicity results of the magneto-electric nanomaterial in example 1, and it can be seen that the material has no toxicity to HEK293 cells and has good biological application potential.
FIG. 9 is a test of the ability of two-dimensional GeH nanoplatelets to scavenge ROS at the cellular level in example 1. In experiments, the cells were first incubated at 1X 10 4 Density of wells/wells was inoculated into 96-well plates and then incubated at 37℃with 5% CO 2 CO of humid air 2 Culturing in an incubator for 24h to allow the cells to adhere to the wall. Following cellular administration, PVP-GeH (10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL) was added at various concentrations to each well, where the concentrations were based on the mass of Ge as a quantitative standard, and incubated for 30min. Then, the cells were treated with 250. Mu. M H 2 O 2 Treated and further incubated at 37℃for 24h. Cells seeded in 96-well plates were incubated with CCK-8 to detect cell viability. Without addition of H 2 O 2 Is considered as a negative control.
Claims (8)
1. The application of the two-dimensional germanium nano-sheet in preparing an AKI disease treatment material related to ROS is characterized in that the two-dimensional germanium nano-sheet has an ultrathin layered structure, and H atoms are covalently modified on the surface of the two-dimensional germanium nano-sheet;
the preparation method of the two-dimensional germanium nanosheets comprises the following steps:
(1) The precursor CaGe with lamellar microstructure is prepared 2 Carrying out rapid heating treatment and quenching treatment, and then immersing in concentrated hydrochloric acid solution for magnetic stirring to obtain a mixed solution;
(2) And centrifuging the obtained mixed solution, removing supernatant, and cleaning precipitate by using anhydrous acetonitrile or acidic deionized water solution to obtain the two-dimensional germanium nanosheets.
2. The use according to claim 1, wherein the two-dimensional germanium nanoplatelets have a lateral dimension of 200-300nm and a thickness of 0.5-2 nm.
3. The use according to claim 1, wherein the two-dimensional germanium nanoplatelets are stable under acidic conditions and degrade under neutral conditions.
4. The use of claim 1, wherein the two-dimensional germanium nanoplatelets further comprise polyvinylpyrrolidone modified on the surface of the two-dimensional germanium nanoplatelets, wherein the mass ratio of polyvinylpyrrolidone to two-dimensional germanium nanoplatelets is 1: (10-20).
5. The use according to claim 1, wherein the temperature of the rapid thermal process is 360-400 ℃; the temperature of the quenching treatment is-196 ℃; the concentration of the concentrated hydrochloric acid solution is 10-13 moL/L.
6. The use according to claim 5, wherein the concentrated hydrochloric acid solution has a concentration of 12moL/L.
7. The use according to claim 1, wherein the magnetic stirring is carried out at a temperature of-40 ℃, at a rotational speed of 500 to 600 revolutions per minute, for a period of 6 to 8 days.
8. The use according to claim 1, wherein the centrifugation is carried out at a speed of 18000 to 20000 rpm for a period of 10 to 30 minutes.
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