CN114917354A - Preparation and application of Cu monatomic nanoenzyme - Google Patents
Preparation and application of Cu monatomic nanoenzyme Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
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- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/34—Copper; Compounds thereof
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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Abstract
The invention discloses a Cu monoatomic nanoenzyme, which is prepared from a compound Cu-N 4 ClG; the preparation method comprises the following steps: step S21, adding the acidified graphene and copper phthalocyanine CuPc into triple distilled water for centrifugation; step S22, carrying out ultrasonic treatment on the product obtained in the step S21; step S23, washing and drying the product obtained in the step S22 to obtain a CuPc functionalized G composite material; step S24, carrying out Ar atmosphere heat treatment on the CuPc functionalized G composite material; step S25, theCooling the product obtained in the step S24, and then placing the product in HCl solution for stirring treatment; step S26, washing and drying to obtain the monoatomic nanoenzyme Cu-N 4 ClG are provided. The Cu-N4ClG monatomic nanoenzyme designed by the application has excellent CAT and SOD-like activities, and provides an effective strategy and a new idea for treating osteoarthritis and chronic diseases related to oxidative stress in the future.
Description
Technical Field
The invention belongs to the field of nano enzyme preparation, and particularly relates to preparation and application of Cu monatomic nano enzyme.
Background
Osteoarthritis is a chronic joint disease, and currently, for treating osteoarthritis, oral nonsteroidal anti-inflammatory drugs (NSAIDs) and drugs for improving local microcirculation are mainly adopted, and meanwhile, hyaluronic acid or corticosteroids are injected into joint cavities. However, these methods only alleviate the symptoms of the patients to various degrees and hardly stop the progression of the disease, which results in the development of osteoarthritis patients to the late stage, which requires a great deal of time and money for treatment and care, and even expensive joint replacement surgery. Therefore, there is an urgent need to develop a new safe and effective osteoarthritis treatment strategy in the field of clinical treatment in orthopedics.
Studies have shown that excessive accumulation of free radicals such as active oxygen in joints is a major cause of osteoarthritis, can cause oxidative stress damage to biofilms, DNA, lipids and proteins, and leads to over-expression of many inflammatory factors, leading to cartilage degradation and osteoarthritis pathogenesis. Strategies based on reactive oxygen free radical scavengers, which can scavenge excess reactive oxygen free radicals and inhibit oxidative stress, have proven to be promising tools for the treatment of osteoarthritis. And is nanoThe enzyme has high activity of catalase-like and superoxide dismutase-like enzymes as a potential active oxygen scavenger, can effectively remove active oxygen free radicals, and is of great interest in osteoarthritis treatment. For example, MnO reported in related research 2 The nano particles, the hollow Prussian blue nano enzyme, the modified ZIF-8 nano particles and the dopamine black pigment nano particles show that the nano particles have a good treatment effect on osteoarthritis. However, the existing nanoenzymes still have a series of problems of strong cytotoxicity, low selectivity, low catalytic activity, low utilization rate of metal atoms and the like, and the performance and further application of the nanoenzymes are severely limited.
Chinese patent CN113457659A discloses a transition metal monoatomic nanoenzyme, which is obtained by heat-treating a transition metal-doped metal-organic framework material prepared using water as a solvent, and a preparation method and use thereof. The prepared transition metal monatomic nanoenzyme has uniform morphology, specific surface area and pore size, has atomically dispersed active sites, and provides a platform for further research on the metal active center and the catalytic mechanism of the monatomic catalyst. Experimental results show that the prepared transition metal monatomic nanoenzyme has good activity of imitating oxidase, peroxidase and halogen peroxidase, can be used for preparing enzyme imitating preparations with high catalytic activity, and has wide application prospects in the fields of antibiosis, tumor resistance, wastewater treatment, immunoblot analysis and the like.
In the research, the applicant found that the utilization rate of the monatomic nanoenzyme can reach as high as 100% because the monatomic nanoenzyme has very excellent utilization rate of metal atoms. Meanwhile, the copper-based metal-loaded nano-enzyme catalyst has strong metal-carrier interaction, a low coordination environment, a clear electronic structure and geometric structure, high selectivity and metal atom distribution stability, the excellent performances enable the copper-based metal-loaded nano-enzyme catalyst to simulate a natural highly-evolved enzyme catalysis center, and a monoatomic enzyme is taken as a design concept of the enzyme, so that the copper-based metal-loaded nano-enzyme catalyst becomes a good substitute of the traditional enzyme. However, how to use Cu to prepare monatomic nanoenzyme to treat osteoarthritis becomes a difficulty of current research.
Disclosure of Invention
In order to solve the problems, a Cu monatomic nanoenzyme is prepared, so that active oxygen is effectively removed, and the technical effect of treating osteoarthritis is achieved.
In order to achieve the effect, the invention designs preparation and application of the Cu monatomic nanoenzyme.
A Cu monoatomic nanoenzyme which is composed of a compound Cu-N 4 ClG is added.
Preferably, the preparation method of the Cu monatomic nanoenzyme comprises the following steps:
step S21, adding graphene and copper phthalocyanine CuPc into triple distilled water for centrifugation;
step S22, carrying out ultrasonic treatment on the product obtained in the step S21;
step S23, washing and drying the product obtained in the step S22 to obtain a CuPc functionalized G composite material;
step S24, carrying out Ar atmosphere heat treatment on the CuPc functionalized G composite material;
step S25, cooling the product obtained in the step S24, and then placing the product in HCl solution for stirring treatment;
step S26, washing and drying to obtain the monoatomic nanoenzyme Cu-N 4 ClG。
Preferably, in the step S21, the mass ratio of graphene to copper phthalocyanine is 1-4: 2 to 10.
Preferably, in the step S22, the ultrasonic treatment time is 1-6 h.
Preferably, in step S23, the washing method includes: repeatedly washing with ethanol;
the drying treatment method comprises the following steps: drying at 40 deg.C under vacuum for 12 h.
Preferably, in step S24, the Ar atmosphere heat treatment method includes: and (3) heating the product obtained in the step S23 to 600-900 ℃ in an Ar atmosphere, and carrying out heat treatment for 1-5 h.
Preferably, in the step S25, the concentration of the HCl solution is 3mol/L HCl solution.
Preferably, in the step S25, the stirring method is to stir for 6-18h at 40-80 ℃.
Preferably, in step S26, the washing and drying method includes: washing until the solution is neutral, and vacuum drying at 60 deg.C for 24 h.
A medicine prepared from Cu monatomic nanoenzyme can be used for treating osteoarthritis.
The application has the advantages and effects as follows:
1. the method comprises the steps of constructing a Cl-N doped graphene loaded Cu monoatomic nanoenzyme Cu-N by taking graphene as a carrier and copper phthalocyanine with a specific Cu coordination environment as a Cu and N source 4 ClG, which have excellent CAT and SOD like activities, can be of great significance in the treatment of osteoarthritis.
2. The application proves that Cu-N is proved through experimental results 4 ClG is effective in removing O 2 ·-、·OH、H 2 O 2 And the like, and has excellent CAT and SOD enzyme activities. At the same time, Cu-N 4 ClG has good biological safety, and can effectively remove active oxygen in chondrocyte in osteoarthritis.
3. The method creatively adjusts the monoatomic copper active center through the accurate coordination of chlorine and nitrogen, and establishes the atomic-scale engineering Cu-N 4 ClG monoatomic nanoenzyme, has excellent CAT and SOD like activities, and is used for treating osteoarthritis; however, no literature and patent reports exist on the preparation method of the graphene-supported Cu monatomic nanoenzyme accurately coordinated by chlorine and nitrogen and the research on applying the graphene-supported Cu monatomic nanoenzyme to OA treatment; the application not only provides scientific basis for further application and expansion of the monatomic nanoenzyme, but also provides effective strategy and new idea for treating osteoarthritis and chronic diseases related to oxidative stress in the future.
The foregoing description is only an overview of the technical solutions of the present application, so that the technical means of the present application can be clearly understood, and the present application can be implemented according to the content of the description, and the foregoing and other objects, features, and advantages of the present application can be more clearly understood.
The above and other objects, advantages and features of the present application will become more apparent to those skilled in the art from the following detailed description of specific embodiments thereof, taken in conjunction with the accompanying drawings.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 shows a Cu-N alloy according to the present invention 4 A ClG SAzyme high resolution TEM image;
FIG. 2 shows a Cu-N alloy according to the present invention 4 ClG ESR measurements with G, evaluating their plot against ROS scavenging ability;
FIG. 3 shows a Cu-N of the present invention 4 ClG and G are subjected to H2O2 decomposition and comparison, and the enzymatic activity of the enzyme on CAT is evaluated;
FIG. 4 shows a Cu-N of the present invention 4 ClG in a CCK-8 experiment with G to evaluate the biocompatibility profile;
FIG. 5 shows a Cu-N alloy according to the present invention 4 ClG DCFH-DA probe detection with G, and fluorescence profiles assessing ROS scavenging ability under different control materials and treatment conditions.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. In the following description, specific details such as specific configurations and components are provided only to help the embodiments of the present application be fully understood. Accordingly, it will be apparent to those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, descriptions of well-known functions and constructions are omitted in the embodiments for clarity and conciseness.
It should be appreciated that reference throughout this specification to "one embodiment" or "the embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present application. Thus, the appearances of the phrase "one embodiment" or "the present embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Further, the present application may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed.
The term "and/or" herein is merely an association relationship describing an associated object, and means that there may be three relationships, for example, a and/or B, which may mean: the three cases of A alone, B alone and A and B together exist, and the term "/and" in this document describes another associated object relationship, which means that two relationships may exist, for example, A/and B, which may mean: the presence of a alone, and both cases a and B alone, and further, the character "/" herein generally means that the former and latter associated objects are in an "or" relationship.
The term "at least one" herein is merely an association relationship describing an associated object, and means that there may be three relationships, for example, at least one of a and B, may mean: a exists alone, A and B exist simultaneously, and B exists alone.
It is further noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion.
Example 1
This example mainly describes a Cu monoatomic nanoenzyme and a method for preparing the same.
A Cu monoatomic nanoenzyme, which is composed of a compound Cu-N 4 ClG is added.
A preparation method of Cu monatomic nanoenzyme comprises the following steps:
step S21, adding graphene and copper phthalocyanine CuPc into triple distilled water for centrifugation;
step S22, carrying out ultrasonic treatment on the product obtained in the step S21;
step S23, washing and drying the product obtained in the step S22 to obtain a CuPc functionalized G composite material;
step S24, carrying out Ar atmosphere heat treatment on the CuPc functionalized G composite material;
step S25, cooling the product obtained in the step S24, and then placing the product into HCl solution for stirring treatment;
step S26, washing and drying to obtain the monoatomic nanoenzyme Cu-N 4 ClG。
Further, in the step S21, the mass ratio of the graphene to the copper phthalocyanine is 1-4: 2 to 10.
Further, in the step S22, the ultrasonic treatment time is 1-6 h.
Further, in step S23, the washing method includes: repeatedly washing with ethanol;
the drying treatment method comprises the following steps: drying at 40 deg.C under vacuum for 12 h.
Further, in step S24, the Ar atmosphere heat treatment method includes: and (3) heating the product obtained in the step S23 to 600-900 ℃ in Ar atmosphere, and carrying out heat treatment for 1-5 h.
Further, in the step S25, the concentration of the HCl solution is 3mol/LHCl solution.
Further, in the step S25, the stirring method is to stir for 6-18h at 40-80 ℃.
Further, in step S26, the method for washing and drying includes: washing until the solution is neutral, and vacuum drying at 60 deg.C for 24 h.
A medicine prepared from Cu monatomic nanoenzyme is used for preparing osteoarthritis.
Example 2
This example mainly describes the preparation of a Cu monatomic nanoenzyme.
According to the invention, 5-20mg of graphene and 10-50mg of copper phthalocyanine (CuPc) are firstly added into a centrifuge tube containing 8mL of tertiary water, ultrasonic treatment is carried out for 1-6h, then repeated washing is carried out by ethanol, and vacuum drying is carried out for 12h at 40 ℃ to obtain the CuPc functionalized G composite material. Then placing the mixture in Ar atmosphere to raise the temperature to 600-900 ℃ (5 ℃/min), and carrying out heat treatment for 1-5 h. After cooling to room temperature, placing the sample in 3mol/LHCl solution, stirring for 6-18h at 40-80 ℃, then washing until the solution is neutral, and drying in vacuum for 24h at 60 ℃ to obtain the N-doped graphene-loaded N and Cl coordinated Cu monatomic nanoenzyme (Cu-N) 4 ClG SAzyme)。
Example 3
Based on the above example 1, this example mainly describes the verification of the effect of a Cu monatomic nanoenzyme in osteoarthritis treatment.
FIG. 1 shows Cu-N 4 High resolution TEM image of ClG nanoenzymes. The TEM images clearly show that the Cu monoatomic atoms are uniformly distributed on the graphene surface, illustrating the formation of Cu monoatomic atoms. At the same time, Cu-N in FIG. 1 4 ClG, only a broad peak around 25 deg. is detected due to the characteristic diffraction of graphitic carbon, and no diffraction peak of Cu nanoparticles is observed, indicating that Cu-N 4 ClG there is no significant aggregation of Cu atoms.
FIG. 2 shows the Cu-N of the prepared material 4 ClG shown in FIG. 2a, using 5-tert-butylcarbonyl-5-methyl-1-pyrroline oxide (BMPO) as a trapping agent and H2O2 as a generator of O2. cndot. -radical, Cu-N was investigated by Electron Spin Resonance (ESR) 4 ClG has effect in scavenging O2.When Cu-N is not added 4 ClG, the ESR signal of O2 is stronger, indicating that there is excess O2, and Cu-N is added 4 ClG, the ESR signal of O2. cndot. -decreased with increasing concentration, and almost completely decreased when the concentration was increased to 150ug/mL, indicating excellent ability to scavenge OH radicals. But with Cu-N 4 ClG, G still has a distinct OHERS signal peak (FIG. 2b), indicating that the addition of Cu monoatomic atoms plays a decisive role in increasing the scavenging ability of O2-free radicals. Similarly, we have also investigated Cu-N 4 ClG eliminating OH. Similarly, we have also investigated Cu-N 4 ClG on O2-scavenging action. As shown in FIG. 2c, Cu-N 4 ClG are also effective in scavenging OH radicals in a concentration dependent manner. When adding 100ug/mLCu-N respectively 4 ClG and G (FIG. 2d), the ESR signal for G remains strong, but in contrast, in Cu-N 4 ClG the ESR signal of OH was almost disappeared, confirming its excellent SOD-like activity.
FIG. 3 shows a Cu-N of the present invention 4 ClG and G, which show the effect of H2O2 decomposition, the Cu-N of the prepared material is shown 4 ClG was effective in concentrating H in a concentration-dependent manner (50, 100, 150, 200ug/mL) 2 O 2 Decomposition into O2 (FIG. 3a), and decomposition O 2 The yield of the method is far higher than that of G and pure H 2 O 2 Panel (fig. 3b), showing excellent CAT-like activity. Supplementing specific concentration scheme with figure
FIG. 4 is a diagram showing the synthesis of Cu-N 4 ClG, the biological safety of the material to the cells is the key of primary concern. First, the Cu-N test was performed using CellCountingKit-8(CCK-8) assay system 4 ClG toxicity to chondrocytes. As shown in FIG. 4, Cu-N 4 ClG and G in the concentration range of 200ug/mL (50, 100, 150, 200ug/mL), the cell activity reaches more than 95%, and no obvious cytotoxicity exists. Supplementing specific concentration scheme with figure
FIG. 5 is a graph using H 2 O 2 Induction of chondrocytes to construct OA in vitro cell model, and then further research on Cu-N by using active oxygen fluorescent probe 2 ', 7' -dichlorodiacetate (DCFH-DA) 4 ClG removing cartilageThe ability of intracellular ROS. The DCFH-DA chemical probe can be oxidized by intracellular ROS, so that green fluorescence is generated, and the ROS content in the cells can be reflected by the strength of the fluorescence. As shown in the figure, H 2 O 2 Stimulation can significantly increase the green fluorescence intensity, indicating that excess ROS are produced. And G + H 2 O 2 Group (100ug/mL) still maintained strong green fluorescence intensity, with H 2 O 2 The green fluorescence content of the groups was close. But when Cu-N is added 4 ClG (100ug/mL), the green fluorescence dropped significantly more than 70%. Thus, it was revealed that Cu-N having excellent CAT-and SOD-like enzyme activities 4 ClG can greatly eliminate ROS in OA chondrocyte.
The method takes graphene as a carrier, and copper phthalocyanine with a specific Cu coordination environment is used as a Cu and N source to construct the Cl-doped N-doped graphene loaded Cu monatin nanoenzyme, so that the enzyme has excellent CAT and SOD-like activities, and is applied to OA treatment in advance. The experimental results prove that Cu-N 4 ClG is effective in removing O 2 ·-、·OH、H 2 O 2 And the like, and has excellent CAT and SOD enzyme activities.
The above description is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the present invention, and various modifications and changes may be made by those skilled in the art. Variations, modifications, substitutions, integrations and parameter changes of the embodiments may be made without departing from the principle and spirit of the invention, which may be within the spirit and principle of the invention, by conventional substitution or may realize the same function.
Claims (10)
1. The Cu monoatomic nanoenzyme is characterized in that the Cu monoatomic nanoenzyme is composed of a compound Cu-N 4 ClG.
2. The method for preparing Cu monatomic nanoenzyme according to claim 1, characterized in that it comprises the following steps:
step S21, adding graphene and copper phthalocyanine CuPc into triple distilled water for centrifugation;
step S22, carrying out ultrasonic treatment on the product obtained in the step S21;
step S23, washing and drying the product obtained in the step S22 to obtain a CuPc functionalized G composite material;
step S24, carrying out Ar atmosphere heat treatment on the CuPc functionalized G composite material;
step S25, cooling the product obtained in the step S24, and then placing the product in HCl solution for stirring treatment;
step S26, washing and drying to obtain the monoatomic nanoenzyme Cu-N 4 ClG。
3. The method for preparing the Cu monatomic nanoenzyme according to claim 2, wherein in the step S21, the mass ratio of the graphene to the copper phthalocyanine is 1-4: 2 to 10.
4. The method for preparing the Cu monatomic nanoenzyme according to claim 2, wherein in the step S22, the ultrasonic treatment time is 1-6 hours.
5. The method of claim 2, wherein in step S23, the washing method comprises: repeatedly washing with ethanol;
the drying treatment method comprises the following steps: drying at 40 deg.C under vacuum for 12 h.
6. The method for preparing Cu monatomic nanoenzyme according to claim 2, wherein in step S24, the Ar atmosphere heat treatment method comprises: and (3) heating the product obtained in the step S23 to 600-900 ℃ in an Ar atmosphere, and carrying out heat treatment for 1-5 h.
7. The method of claim 2, wherein in step S25, the HCl solution concentration is 3mol/L HCl solution.
8. The method for preparing the Cu monatomic nanoenzyme of claim 2, wherein in the step S25, the stirring method is stirring at 40-80 ℃ for 6-18 h.
9. The method for preparing the Cu monatomic nanoenzyme according to claim 2, wherein in the step S26, the washing and drying method comprises: the solution is washed until neutral and dried under vacuum at 60 ℃ for 24 h.
10. Use of a Cu monatin nanoenzyme according to claim 1 in the preparation of a medicament for osteoarthritis.
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