CN114890385A - Efficient antioxidant two-dimensional hydrogermanium alkene nanosheet and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a high-efficiency antioxidant two-dimensional hydrogermylene nanosheet and a preparation method and application thereof. The two-dimensional germanium nanosheet has an ultrathin lamellar structure, and H atoms are covalently modified on the surface.
Description
Technical Field
The invention relates to an ultrathin germanium nanosheet (Germanene) with excellent oxidation resistance, uniform size and high specific surface area and a hydrogen (H) atom connected to the surface, a preparation method thereof and related application of the ultrathin germanium nanosheet 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 anion (O) 2 ·- ) Hydrogen peroxide (H) 2 O 2 ) Singlet oxygen (a) 1 O 2 ) And hydroxyl radical (. OH), play an important role in regulating various physiological functions of organisms, participate in mechanisms necessary for growth, health or aging of organisms, and often play an important role as a signaling molecule. There are also potential toxic effects of ROS, including oxidation and damage to DNA, RNA, proteins and membranes, which require multiple antioxidant systems within the cell to maintain ROS at essentially non-toxic levels, and any deviation from this balance can lead to disease development (Trends in plant Science,2016,22(1), 11-19). Acute Kidney Injury (AKI), a disease associated with oxidative stress, clinically results in high mortality rates each year (adv. Although 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 variety of functional Nano-drugs are designed by the majority of researchers, and are used for the anti-oxidation treatment of AKI, such as Black Phosphorus (BP) (Nano Lett.2020,20,1447- 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 ultra-thin germanium nanosheet with excellent oxidation resistance, and a preparation method and applications thereof.
In one aspect, the invention provides a two-dimensional germanium nanosheet, which has an ultrathin layered structure and a surface covalently modified with H atoms (i.e., germane-H, two-dimensional GeH nanosheet).
In the present disclosure, the two-dimensional GeH nanosheets with ultrathin lamellar structure have excellent antioxidant capacity and can scavenge a series of representative Reactive Oxygen Species (ROS), including hydrogen peroxide (H) 2 O 2 ) Superoxide anion (O) 2 ·- ) And hydroxyl radical (. OH), etc., can be well applied to the treatment of AKI diseases related to ROS.
Preferably, the two-dimensional germanium nanosheet has a transverse dimension of 200-300nm and a thickness of 0.5-2 nm.
Preferably, the two-dimensional germanium nanosheets have a PH-responsive degradation behavior, and can be stable in a solution tending to acidity (PH < 7, inflammatory microenvironment) for a longer period of time, the stability time increases with decreasing PH, the two have a linear relationship, and the two rapidly degrade in a neutral state (PH 7.4, normal tissue microenvironment). The selective degradation characteristic can realize the aim of targeted treatment of inflammation and has better biological safety.
Preferably, the two-dimensional germanium nanosheet further comprises polyvinylpyrrolidone modified on the surface of the two-dimensional germanium nanosheet, and the mass ratio of the two-dimensional germanium nanosheet to the polyvinylpyrrolidone is 1: (10-20). Through surface modification of PVP, in-vivo biological application of the material can be better realized.
On the other hand, the invention also provides a preparation method of the two-dimensional germanium nanosheet, which comprises the following steps:
(1) the precursor CaGe with a layered microstructure 2 Carrying out rapid heating treatment and rapid cooling treatment, and then soaking the mixture into concentrated hydrochloric acid solution for magnetic stirring to obtain mixed solution;
(2) and centrifuging the obtained mixed solution, removing supernatant, and cleaning the precipitate by using anhydrous acetonitrile or an acidic deionized water solution to obtain the two-dimensional germanium nanosheet.
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 10-13 moL/L, and preferably 12 moL/L.
Preferably, the temperature of the magnetic stirring is-40 ℃, the rotating speed is 500-600 r/min, 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 rotation speed of the centrifugal treatment is 19000 r/min, and the time is 15-30 min.
In another aspect, the invention further provides an application of the two-dimensional germanium nanosheet in preparation of an AKI disease treatment material related to ROS.
Has the advantages that:
the preparation method disclosed by the invention is simple in preparation process, high in feasibility, low in cost, high in yield, controllable in size of the obtained nano material, excellent in performance and capable of being used as an excellent antioxidant for ROS (reactive oxygen species) scavenging related organisms.
In the nanomaterial system, the H atoms connected to the surfaces of the GeH nanosheets can effectively remove Reactive Oxygen Species (ROS), so that the purpose of relieving Acute Kidney Injury (AKI) is achieved.
Drawings
Fig. 1 is a flow diagram of two-dimensional GeH nanoplates prepared in example 1;
FIG. 2 shows a precursor CaGe for two-dimensional GeH nanosheet synthesis in example 1 2 Can be seen to have a distinct layered structure;
FIG. 3 is a TEM image of two-dimensional GeH nanosheets of example 1, the nanosheets being uniform in size and well dispersed (scale bar, 200 nm);
FIG. 4 is an Atomic Force Microscope (AFM) image of two-dimensional GeH nanoplates of example 1, demonstrating that the nanoplates have an ultra-thin structure, with a thickness of around 1 nm;
FIG. 5 is an infrared absorption spectrum of a two-dimensional GeH nanosheet of example 1, confirming the presence of covalently bonded hydrogen atoms on the nanosheet surface;
FIG. 6 is a Raman spectrum of a two-dimensional GeH nanosheet of example 1, confirming the presence of covalently bonded hydrogen atoms on the nanosheet surface;
FIG. 7 shows that the two-dimensional GeH nanosheets in example 1 generate hydroxyl radicals (. OH) and superoxide anions (O) under certain conditions in an aqueous oxygen environment 2 ·- ) The Electron Spin Resonance (ESR) test results before and after the reaction prove that the two-dimensional GeH nanosheet can effectively eliminate ROS, and is possibly applied to treatment of ROS-related inflammatory diseases;
fig. 8 is a cytotoxicity test result of the two-dimensional GeH nanosheet in example 1, which proves that the material is non-toxic to human embryonic kidney 293(HEK 293) cells and has good biological application potential;
FIG. 9 shows two-dimensional GeH nanosheet pair and H in example 1 2 O 2 Protection of co-incubated treated HEK293 cells.
Detailed Description
The present invention is further illustrated by the following examples, which are to be construed as merely illustrative, and not a limitation of the present invention.
In the disclosure, the surface of the two-dimensional GeH nanosheet modified with hydrogen atoms not only can consume ROS due to the hydrogen atoms connected to the surface of the two-dimensional GeH nanosheet, but also can react with ROS due to the reduction characteristic of Ge, so that the purpose of removing the ROS is achieved. Meanwhile, the two-dimensional GeH nanosheets are used as a nano-drug carrier material, and the high specific surface area of the two-dimensional GeH nanosheets greatly improves the ROS removing efficiency, so that the high-efficiency and low-toxicity treatment effect is realized.
In the invention, the preparation method of the two-dimensional GeH nanosheet is simple and feasible in synthesis process and controllable and accurate in reaction conditions. Fig. 1 is a flowchart of preparing a two-dimensional GeH nanosheet according to an embodiment of the present invention. The following is an exemplary description of a method for preparing a two-dimensional GeH nanosheet having a surface modified with hydrogen atoms.
Mixing Ca block and Ge powder according to a stoichiometric ratio of 1: 2, weighing, and uniformly mixing and putting into a quartz tube plated with a carbon film. Then, while vacuumizing, the tube sealing treatment is carried out by using oxyhydrogen flame. Putting the quartz tube filled with the material 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 。
Adding a precursor CaGe 2 Grinding into powder to obtain powder CaGe 2 。
Mixing powdered CaGe 2 Subjected to a thermal expansion treatment (for example heating to 380 ℃), and then immersed in liquid N 2 (L-N 2 ) From medium to L-N 2 Complete gasification (e.g. quenching to-196 ℃) to give the precursor CaGe with a layered microstructure 2 And (3) powder.
The precursor CaGe with a layered microstructure 2 Immersing the powder in concentrated hydrochloric acid solution, magnetically stirring at low temp, and adding HCl and CaGe 2 Reaction takes place to generate CaCl 2 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 reaction by-products) was removedSubstance CaCl 2 Etc.), collecting the precipitate and washing the precipitate for multiple times (for example, three times) with an aqueous acetone or acidic deionized water solution to obtain the two-dimensional GeH nanosheet.
According to the invention, the ultrathin two-dimensional GeH nanosheet which is good in stability, uniform in size, safe and low in toxicity is synthesized by the simple, feasible 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.
According to the invention, the obtained two-dimensional GeH nanosheet can stably exist in a weak acid microenvironment of inflammation, and is easy to degrade in a neutral condition of a normal tissue. So that the inflammation treatment has targeting property, the biological safety is improved, and the possibility of systemic toxicity caused by systemic distribution is avoided. Meanwhile, Ge can react with ROS due to the reducibility of Ge, reduce the oxidative stress level of inflammation position, and the hydrogen atom connected with the surface of Ge further enhances the reaction activity, thus achieving the high-efficiency inflammation treatment effect.
The present invention will be described in detail by way of examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings. The specific process parameters and the like of the following examples are also merely one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.
Example 1
Preparing a two-dimensional GeH nano sheet: 1.092g of Ca block and 3.919g of Ge powder were weighed, mixed uniformly and put into a carbon film-coated quartz tube. Under the condition of fully vacuumizing, the tube is sealed by using oxyhydrogen flame. Putting the quartz tube subjected to tube sealing operation into a muffle furnace, heating to 1000 ℃ at a heating rate of 10 ℃/min, annealing for 18h under the condition, and naturally cooling to room temperature to obtain the blocky CaGe 2 . The obtained bulk CaGe 2 Further grinding into powder, and weighing 1g of CaGe powder 2 Again subjected to thermal expansion treatment(heated to 380 ℃ C.), and subsequently immersed in liquid N 2 (L-N 2 ) From medium to L-N 2 Completely gasified (quenched to-196 ℃), and the precursor CaGe with a layered microstructure is obtained 2 And (3) powder. The precursor CaGe with a layered microstructure 2 The powder was immersed in 100ml of concentrated hydrochloric acid solution (12moL/L) and magnetically stirred at-40 ℃ and 600rpm for 24 hours. And then centrifuging at a high speed (19000rpm for 20min), collecting the precipitate, and cleaning the precipitate for three times by using an anhydrous acetone or acidic deionized water solution to obtain the product GeH nanosheet. Further, 1mg (dissolved in 5mL of ethanol) of GeH was mixed with 10mg of PVP (dissolved in 5mL of ethanol), followed by stirring in a water bath at 50 ℃ for 6 hours (500 rpm), and finally the precipitate was collected by centrifugation to obtain PVP-modified GeH (PVP-GeH).
FIG. 2 shows a CaGe precursor of a two-dimensional GeH nanosheet in example 1 2 SEM picture of (1) visually displaying CaGe 2 Has a layered microstructure, and illustrates a precursor CaGe 2 The Ca layer and the Ge layer are alternately arranged by the close interlayer acting force to form the layered compound.
Fig. 3 shows that the two-dimensional GeH nanosheet synthesized in example 1 has a weak bonding force between the inner layers of the precursor in fig. 2 due to reduction by rapid cooling and rapid heating, and the Ca layer can be effectively removed by further concentrated hydrochloric acid treatment, so that the Ge layer with good dispersibility and uniform size is retained.
FIG. 4 is an Atomic Force Microscope (AFM) image of the two-dimensional GeH nanosheet of example 1, showing that it is about 1nm thick, has an ultra-thin structure, and conforms to the characteristics of the nanosheet.
Fig. 5 is an infrared absorption spectrum of the two-dimensional GeH nanosheet in example 1, which confirms successful implementation of covalent modification of hydrogen atoms on the surface of the nanosheet, and synthesis of the GeH nanosheet.
Fig. 6 is a raman spectrum of the two-dimensional GeH nanosheet in example 1, which confirms that covalent modification of hydrogen atoms on the surface of the nanosheet is successfully achieved, and the GeH nanosheet is synthesized.
FIG. 7 shows that the two-dimensional GeH nanosheets in example 1 generate hydroxyl radicals (. OH) and superoxide anions (O) under certain conditions in an aqueous oxygen environment 2 ·- ) The results of Electron Spin Resonance (ESR) tests before and after the reaction,the two-dimensional GeH nanosheet can effectively eliminate ROS.
The cytotoxicity test of the GeH nanosheets in this example 1 was evaluated using a classical CCK-8 kit. In carrying out the CCK-8 experiment, cells were first plated at 1X 10 4 Density of/well into 96-well plates and then 5% CO at 37 ℃ 2 CO of humid air 2 The cells were allowed to adhere to the wall for 24h in an incubator. The adherent cells were then replaced with fresh medium containing PVP-GeH (40. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL, where concentrations are based on Ge quality) in varying concentrations and incubation continued for 24 h. After the incubation was completed, the culture medium was removed and washed 3 times with fresh culture medium. Adding ten times of CCK-8 solution diluted with culture medium into each well, placing at 37 deg.C and containing 5% CO 2 CO of humid air 2 The incubator was incubated for 4 h. Finally, the absorbance (λ 450nm) was measured on a microplate reader. The cytotoxicity index is expressed as a percentage of the cell viability after treatment of the sample relative to the cell viability of the untreated blank. Fig. 8 is a graph showing the cytotoxicity results of the magnetoelectric nanomaterial in this example 1, which shows 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 carrying out the experiment, cells were first plated at 1X 10 4 Density of each well was plated in 96-well plates and then incubated at 37 ℃ with 5% CO 2 CO of humid air 2 The cells were allowed to adhere to the wall for 24h in an incubator. Following cell administration, different concentrations of PVP-GeH (10. mu.g/mL, 5. mu.g/mL, 2.5. mu.g/mL, where concentrations are based on Ge mass) were added to each set of wells and incubated for 30 min. Then, the cells were plated at 250. mu. M H 2 O 2 Treated and further incubated at 37 ℃ for 24 h. Cells seeded in 96-well plates were incubated with CCK-8 to examine cell viability. Without addition of H 2 O 2 The wells of (a) were considered negative controls.
Claims (9)
1. A two-dimensional germanium nanosheet is characterized in that the two-dimensional germanium nanosheet has an ultrathin layered structure, and H atoms are covalently modified on the surface.
2. Two-dimensional germanium nanosheets according to claim 1, wherein the two-dimensional germanium nanosheets have a lateral dimension of 200 to 300nm and a thickness of 0.5 to 2 nm.
3. Two-dimensional germanium nanoplatelets according to claim 1 or 2 wherein the two-dimensional germanium nanoplatelets are stable in acidic conditions and degrade in neutral conditions.
4. Two-dimensional germanium nanosheets according to any one of claims 1 to 3, further comprising polyvinylpyrrolidone modified at the surface of the two-dimensional germanium nanosheets, the mass ratio of polyvinylpyrrolidone to two-dimensional germanium nanosheets being 1: (10-20).
5. A method of preparing two-dimensional germanium nanoplates as described in any of claims 1-4, comprising:
(1) the precursor CaGe with a layered microstructure 2 Carrying out rapid heating treatment and rapid cooling treatment, and then soaking the mixture into concentrated hydrochloric acid solution for magnetic stirring to obtain mixed solution;
(2) and centrifuging the obtained mixed solution, removing supernatant, and cleaning the precipitate by using anhydrous acetonitrile or an acidic deionized water solution to obtain the two-dimensional germanium nanosheet.
6. The preparation method according to claim 5, wherein the temperature of the rapid heat treatment is 360-400 ℃; the temperature of the quenching treatment is-196 ℃; the concentration of the concentrated hydrochloric acid solution is 10-13 moL/L, and preferably 12 moL/L.
7. The preparation method according to claim 5 or 6, wherein the temperature of the magnetic stirring is-40 ℃, the rotating speed is 500-600 r/min, and the time is 6-8 days.
8. The method according to any one of claims 5 to 7, wherein the centrifugation is performed at a rotation speed of 18000 to 20000 rpm for 10 to 30 minutes.
9. Use of two-dimensional germanium nanoplates as described in any of claims 1-4 in the preparation of a material for the treatment of an AKI disease associated with ROS.
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