CN-117064910-B - Composite nano enzyme and preparation method and application thereof

Abstract

The invention discloses a composite nano enzyme, a preparation method and application thereof, wherein the composite nano enzyme takes Prussian blue nano particles as a carrier, a phospholipid bilayer formed by dimyristoyl phosphatidylcholine is coated on the surfaces of the Prussian blue nano particles, and (2.3-dioleoyl-propyl) -trimethylamine and undecylimidazole are inlaid in the phospholipid bilayer. The invention discloses a composite nano-enzyme formed by modifying DOTAP and imidazole groups on the surfaces of Prussian blue nano-particles for the first time, the obtained composite nano-enzyme can effectively catalyze ester hydrolysis and at least generate two orders of magnitude acceleration, which provides possibility for directly hydrolyzing cholesterol esters in atherosclerosis plaques by nano-drugs, and the invention expands the enzyme-like activity of Prussian blue nano-enzyme through special modification and provides a scheme for simultaneously realizing the multifunctional composite nano-enzyme with ester hydrolase functions, hydrogen peroxidase-like, superoxide dismutase-like characteristics and the like.

Inventors

• ZHANG YU
• Yu Puge
• WANG JIAMING
• MA MING

Assignees

• 东南大学

Dates

Publication Date

20260505

Application Date

20230920

Claims (7)

1. 1. The composite nano enzyme is characterized in that Prussian blue nano particles are used as carriers, a phospholipid bilayer formed by dimyristoyl phosphatidylcholine is coated on the surfaces of the Prussian blue nano particles, and (2, 3-dioleoyl-propyl) -trimethylamine and undecylimidazole are inlaid in the phospholipid bilayer.
2. 2. A method for preparing the composite nano enzyme according to claim 1 is characterized by comprising the steps of steaming a mixed acetonitrile solution of dimyristoyl phosphatidylcholine, (2, 3-dioleoyl-propyl) -trimethylamine and undecylimidazole in a rotary mode, adding Prussian blue nanoparticle aqueous solution, and purifying to obtain the composite nano enzyme, wherein the concentration ratio of dimyristoyl phosphatidylcholine, (2, 3-dioleoyl-propyl) -trimethylamine and undecylimidazole in the mixed acetonitrile solution is 10 (5-10): 5-10, the volume ratio of dimyristoyl phosphatidylcholine, (2, 3-dioleoyl-propyl) -trimethylamine and undecylimidazole in the mixed acetonitrile solution is 975 (25-50), and the volume ratio of the mixed acetonitrile solution and Prussian blue nanoparticle aqueous solution is 1050 (300-400).
3. 3. The method according to claim 2, wherein the preparation method of the Prussian blue nanoparticle comprises the steps of dissolving FeCl 3 •6H 2 O in an aqueous solution of polyvinylpyrrolidone, dropwise adding an aqueous solution of K 4 [Fe(CN) 6 ]•3H 2 O, and purifying to obtain the Prussian blue nanoparticle.
4. 4. The method according to claim 3, wherein the mass-to-volume ratio of FeCl 3 •6H 2 O to polyvinylpyrrolidone aqueous solution is (25-30) 80mg/mL.
5. 5. The method according to claim 3, wherein the dropping rate of the K 4 [Fe(CN) 6 ]•3H 2 O aqueous solution is 30-50 mL/h.
6. 6. The method of claim 3, wherein the molar ratio of FeCl 3 •6H 2 O to K 4 [Fe(CN) 6 ]•3H 2 O is (3.5-4.5): 1.
7. 7. Use of the complex nanoenzyme of claim 1 for the preparation of a reagent or medicament for hydrolysing Cbz-Phe-Onp.

Description

Composite nano enzyme and preparation method and application thereof Technical Field The invention relates to a composite nano-enzyme and a preparation method and application thereof, belonging to the field of biomedicine. Background In the process of atherosclerosis, plaque rupture causes thrombosis, which is a main cause of serious diseases such as coronary heart disease, cerebral infarction and the like. Wherein foam cells developed from macrophages accumulate Cholesterol Esters (CE) in large amounts and protrude into the lumen of the vessel wall to form plaques, while lipid-rich plaques are prone to rupture. In cells, (neutral) cholesterol esterases (nCEH) are responsible for hydrolyzing cholesterol esters to free cholesterol. Intracellular cholesterol esters can be transported out of the cell only by nCEH degradation to free cholesterol, and hydrolysis of nCEH is therefore considered a key step in reverse cholesterol transport. NCEHs has the main effects of hydrolyzing cholesterol ester into cholesterol and fatty acid, so as to promote the outflow of cholesterol, reduce the accumulation of cholesterol ester in cells, and simultaneously has better effects of nCEHs in inhibiting inflammation and the like. Therefore, in the atherosclerosis cells, the high-expression cholesterol hydrolase can promote CE hydrolysis and intracellular free cholesterol excretion, inhibit foam cell development and plaque formation, effectively delay and inhibit atherosclerosis lesions, and be beneficial to preventing plaque development and disease burst effects such as coronary heart disease, cerebral infarction and the like, and inhibit disease development from the source. Nanoenzymes are important leading research directions in the biomedical nanotechnology field, and are also important progress and innovation in the enzyme engineering field. Compared with natural enzymes, the nano-enzyme has the advantages of simple preparation, large-scale production, strong environmental tolerance, low preparation and storage cost, reusability and the like. In the field of artificial hydrolases, short peptide-gold, carbon nanocomposites and metal micelles have been demonstrated to have a lipid hydrolyzing effect many times. The gold nano-composite is characterized in that gold nano-particles are connected with related alkyl groups with special groups by using Au-S bonds, and then the gold nano-particles are self-assembled with short peptides to form a final composite, the groups carried on the short peptides of the composite play a role in hydrolysis, wherein partial multi-group synergism is utilized, and partial local microenvironment created by gold nano-particles is utilized to further accelerate catalysis. The carbon nano-composite is similar to the carbon nano-composite, gold nano-particles are replaced by rigid carbon nano-materials, and meanwhile, the carbon nano-materials can create a hydrophobic microenvironment to promote proton transfer and improve catalytic activity. The metal micelle combines the functional ligand with the ion complex to form colloid, which contains catalytic center and simulates hydrophobic microenvironment, and has strong hydrolytic property to ester. In the above and other artificial imitation ester hydrolases, the main group that plays a key role in hydrolysis is the imidazole group. The active centers of ester hydrolases such as cholesterol ester hydrolase contain histidine, and the imidazole group carried by the histidine is the site for hydrolysis. In the catalytic triad, the imidazole groups also play a role in catalytic hydrolysis as an important part. Meanwhile, in the construction process of most artificial hydrolytic enzymes, a local microenvironment is created by utilizing a metal or other material carrier so as to simulate natural enzymes, and the catalytic activity is improved. Currently, the main clinical approach to atherosclerosis is cholesterol lowering drugs, but there is no effective method to directly lower the lipid content in plaque, and no nano-drug based on hydrolyzed cholesterol esters in plaque. Prussian blue nano-enzyme has Catalase (CAT) like and superoxide dismutase (SOD) like functions, and has been proved to play a role in regulating inflammatory microenvironment and relieving atherosclerosis in plaques. At present, whether the Prussian blue nano-enzyme has hydrolytic activity is not reported, so that the Prussian blue nano-enzyme is not used for preparing the composite nano-enzyme with the characteristic of ester hydrolytic enzyme to treat atherosclerosis. Disclosure of Invention The invention aims to solve the technical problem of providing a composite nano-enzyme with ester hydrolase characteristics, and a preparation method and application thereof. According to the technical scheme, the composite nano-enzyme is provided, prussian blue nano-particles are used as carriers, a phospholipid bilayer formed by dimyristoyl phosphatidylcholine is coated on the surfaces of the Prussi