Single-chain peptide folded nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a single-chain peptide folded nano-enzyme as well as a preparation method and application thereof, and belongs to the field of medical medicine materials. The skeleton selected by the invention is single-chain polyamino acid, and the nanoparticles formed by crosslinking can simulate folding of protein in morphology and can be degraded in vivo. A high-molecular polymer methoxyl-polyethylene glycol-amino group is used as an initiator, a middle segment is polyamino acid with two carboxyl groups or polyamino acid with two amino groups, and carboxyl / amino groups are coordinated with transition metal to form a relatively stable hammer cuttage structure. Different enzyme activities can be simulated after different metals are used for crosslinking. Wherein the peroxidase-like and oxidase-like nanoparticles can generate oxidative stress, and the superoxide dismutase-like and catalase-like nanoparticles can remove active oxygen. And a hydrophilic side chain is selected at the tail segment to form the hydrophilic chain-sphere-chain nano enzyme. The single-chain peptide folded nano-enzyme prepared by the invention can be applied to multiple fields such as anti-tumor and anti-inflammatory activity research or preparation of related drugs according to different enzymatic activities.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical drug materials, and in particular relates to a single-chain peptide folding nanozyme, a preparation method thereof, and applications in anti-tumor and anti-inflammatory activity research or related drug preparation. Background Art

[0002] Enzymes are specific and efficient biocatalysts involved in most biocatalytic reactions. Artificial enzymes have emerged to address the inherent shortcomings of natural enzymes, such as volatility, high cost, and laborious preparation. Initially, the combination of enzyme / polymer / metal particles was called nanozyme, where the polymer encapsulates the enzyme to the desired location, where it exerts its enzymatic activity. Subsequently, nanomaterials with enzyme-like properties have become widely referred to as nanozymes. Since 2007, when Academician Yan Xiyun's group proposed that Fe₃O₄ exhibits horseradish peroxidase-mimicking activity, various nanoparticles, such as iron oxide, graphene, fullerenes, and MOF-derived nanomaterials, have been extensively studied as enzyme-like substances. While these inorganic nanozymes mimic the production and scavenging of reactive oxygen species by natural enzymes, they lack the structural compatibility of the flexible, dynamic, and folded natural enzymes. Furthermore, the chemical materials used are non-degradable in the body, leading to residual toxicity during metabolism. Summary of the Invention

[0003] In response to the above problems, the present invention provides a single-chain peptide folding nanozyme, a preparation method thereof, and its application in anti-tumor, anti-inflammatory activity research or related drug preparation. The single-chain peptide folding nanozyme has two hydrophilic segments and a middle segment of polyglutamic acid, which can coordinate with metals to fold to form enzyme-mimicking nanoparticles. The artificial polypeptide synthesized by NCA ring-opening polymerization has functional groups that do not exist in nature, and contains monomers with various functional groups, which are randomly polymerized into single-chain polyamino acids. The present invention overcomes the defects of inorganic nanozymes. Polyamino acids coordinate and connect metals. Nanoparticles of peroxidase (POD) and oxidase (OXD) can be used for anti-tumor activity research and the preparation of related drugs. Nanoparticles of catalase (CAT) and superoxide dismutase (SOD) can be used for anti-inflammatory activity research and the preparation of related drugs.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] One of the purposes of the present invention is to provide a method for preparing a single-chain peptide folding nanozyme, the method comprising the following steps:

[0006] (a) Synthesizing a side chain having a group that can be hydrolyzed into a carboxyl group - NCA (N-carboxyl cyclic anhydride) monomer and an amino acid - NCA (N-carboxyl cyclic anhydride) monomer having a hydrophilic group on the side chain (e.g. Figure 1 );

[0007] (b) preparing a single-chain triblock polymer comprising an initiator segment, a middle segment, and a terminal segment by sequential ring-opening polymerization; wherein the middle segment is a group-NCA polymer that can be hydrolyzed to a carboxyl group, and the terminal segment is a hydrophilic amino acid-NCA polymer;

[0008] (c) hydrolyzing the groups in the middle segment that can be hydrolyzed into carboxyl groups into carboxyl groups to form a single-chain polyamino acid polymer having a polyamino acid chain as the middle segment;

[0009] (d) The carboxyl group is coordinated and complexed with a transition metal ion to fold the middle segment of the polymer, and then the complexed metal ions are converted into corresponding metal oxides and hydroxides by coprecipitation. The resulting enzyme-mimicking nanoparticles are the single-chain peptide folded nanozyme.

[0010] Preferably, in the step (a), the side chain has a group that can be hydrolyzed to form a carboxyl group - in the NCA monomer, the group that can be hydrolyzed to form a carboxyl group is any one or more of a methyl ester group, an ethyl ester group, a tert-butyl ester group, an n-butyl ester group, a benzyl group, a substituted benzyl group, a diphenylmethyl ester group, an amide group, a hydrazide group, a potassium salt, a sodium salt and a triethylamine salt; the NCA parent amino acid is any one or more of aspartic acid, glutamic acid, lysine and arginine.

[0011] More preferably, the group that can be hydrolyzed to form a carboxyl group is a tert-butyl ester group.

[0012] Preferably, in the amino acid-NCA monomer having a hydrophilic group on the side chain in step (a), the hydrophilic group includes an ethylene glycol oligomer, and the ethylene glycol oligomer is any one or more of monoethylene glycol, diethylene glycol and triethylene glycol; the NCA parent amino acid is any one or more of aspartic acid, glutamic acid, lysine and arginine.

[0013] More preferably, the amino acid-NCA monomer having a hydrophilic group on the side chain may be any one of ethylene glycol-glutamic acid-NCA, ethylene glycol-cysteine-NCA, ethylene glycol-lysine-NCA and ethylene glycol-serine-NCA.

[0014] More preferably, the ethylene glycol oligomer is diethylene glycol, and the amino acid is glutamic acid, that is, the amino acid-NCA monomer having a hydrophilic group on the side chain is diethylene glycol-glutamic acid-NCA.

[0015] Preferably, the initiating segment in step (b) is an amino group and the other segment is a methoxy group, which can be any one or more of polyethylene, polystyrene, methoxy-polyethylene glycol-amino, polymethyl methacrylate, polyacetic acid lactone, polylactic acid and polyethylene glycol monomethyl ether.

[0016] More preferably, the initiating segment is methoxy-polyethylene glycol-amino.

[0017] Preferably, the hydrolysis reagent in step (c) is any one or more of acetic acid, hydrochloric acid, hydrogen bromide, potassium hydroxide, sodium borohydride, trifluoroacetic acid and boron trifluoride diethyl ether; the number average molecular weight of the single-chain polyamino acid is 100-3,000,000.

[0018] More preferably, the hydrolysis reagent is trifluoroacetic acid; the number average molecular weight of the single-chain polyamino acid is 500-50,000.

[0019] Preferably, the metal ion used for coordination cross-linking in step (d) is any one or more of Fe 2+ , Fe 3+ , Cu 2+ , Zn 2+ , Mn 2+ , Mn 3 + , Mn 4+ , Co 2+ , Co 3+ , Cr 2+ , Cr 3+ , Pt 2+ , Pt 4+ , Au + , Au 3+ , Ag + , Ca 2+ ; the precipitant used for co-precipitation includes a base, hydrogen sulfide and / or a sulfide salt, wherein the base is any one or more of NH3·H2O, NaOH, KOH, Ca(OH)2, CsOH, and the sulfide salt is any one or more of Na2S, K2S, Cs2S.

[0020] More preferably, the metal ion used for coordination cross-linking is a mixture of Fe 2+ , Fe 3+ .

[0021] Preferably, the solvent used for polyamino acid biomimetic folding in step (d) is a solvent capable of dissolving three segments, specifically any one or more of dimethyl sulfoxide (DMSO), dichloromethane (DCM), water (H2O), 3,3'-dichlorobenzidine (DCB), dimethylacetamide (DMA), ethylene glycol, aniline, acetic acid, nitromethane, pyridine, dioxane and dimethyl phthalate.

[0022] More preferably, the solvent used for polyamino acid biomimetic folding is water (H2O).

[0023] The metal used for forming the enzyme-mimicking nanoparticles in the present application can be selected from iron, manganese, cobalt, copper, zinc, gold, magnesium and the like. Preferably, it is iron.

[0024] The enzymes that can be simulated in the present application are peroxidase, superoxide dismutase, catalase and oxidase. One / more than one enzyme activity is selected according to different metals.

[0025] The second object of the present application is to provide a single-chain peptide folding nanozyme prepared by any of the above preparation methods.

[0026] The third object of the present application is to provide the use of the single-chain peptide folding nanozyme in the preparation of an antitumor drug and / or an anti-inflammatory drug, wherein the single-chain peptide folding nanozyme with peroxidase-like (POD) and / or oxidase-like (OXD) activity is used for the preparation of an antitumor drug; the single-chain peptide folding nanozyme with superoxide dismutase-like (SOD) and / or catalase-like (CAT) activity is used for the preparation of an anti-inflammatory drug.

[0027] The fourth object of the present application is to provide a medicine containing the single-chain peptide folding nanozyme.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] (1) The amino acids selected in the present application have good biocompatibility, can directly initiate the preparation of polyamino acids, the conditions are relatively easy to meet, and the hydrolysis is relatively easy and not disturbed. The single-chain peptide folding nanozyme of the present application is polyamino acid in structure and is degradable in vivo, and has higher safety.

[0030] (2) The single-chain polyamino acid in the present application has smaller size compared with multi-chain polyamino acid, can simulate the folding of proteins to occur intramolecular crosslinking, and has more specific reaction with other functional groups. The "chain-ball-chain" single-chain peptide folding nanozyme of the present application can fit the natural folding of natural enzymes, and the grafting structure is more stable.

[0031] (3) The single-chain peptide folding nanozyme in the present application has small size, only 5-25 nm, has the effects of easily crossing physiological barriers and entering human organs that macromolecules cannot enter to play a drug effect. It has good water solubility and can easily move between cells. The carboxyl group in the middle section can also be connected with drugs / fluorescent dyes to further broaden the application. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The synthesis schematic diagram of the monomer with side chain having hydrolyzable group -NCA (N-carboxyl cyclic anhydride) and the amino acid monomer with hydrophilic group -NCA (N-carboxyl cyclic anhydride) in the present application;

[0033] Figure 2 The reaction schematic diagram in step one in the embodiment of the present application;

[0034] Figure 3Reaction scheme for step two in the present embodiment;

[0035] Figure 4 Reaction scheme for step three in the present embodiment;

[0036] Figure 5 Reaction scheme for step four in the present embodiment;

[0037] Figure 6 Reaction scheme for step five in the present embodiment;

[0038] Figure 7 ESR spectrum of hydroxyl radical generation in Example 1 and Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0039] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or substitutions of the method, step or condition of the present application, without departing from the spirit and the essence of the present application, are within the scope of the present application. The reagents and instruments used in the following examples are commercially available, and the methods used in the examples are consistent with the commonly used methods, unless otherwise specified.

[0040] In the present application, the sources of the raw materials used are shown in Table 1, and the sources of the instruments are shown in Table 2.

[0041] Table 1 Sources of raw materials

[0042]

[0043]

[0044] Table 2 Sources of instruments

[0045] Device Name Specifications Origin Rotary Evaporator R-215 BUCHI vacuum drying oven DZF-6020 Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory centrifuge Centrifuge5430R eppednorf magnetic stirrer RCTBS25 IKA glove box SG1200 / 750TS Weige Gas Purification Technology (Suzhou) Co., Ltd. freeze dryer LGJ-12A Sihuan Furui Technology Development (Beijing) Co., Ltd.

[0046] The technical solutions of the present application are further described in detail below in combination with examples.

[0047] Example 1

[0048] The present embodiment provides a single-chain peptide folding nanozyme, and the preparation method is as follows:

[0049] First step Figure 2 ):

[0050] Triphosgene (3.6 g, 13.28 mmol, phosgene / γ-tert-butyl-L-glutamic acid = 2.74 / 1) and 78 mL of ultra-dry THF were stirred in a tomato flask under nitrogen. The reactor was kept constant at 20°C and the mixture was stirred until the triphosgene was completely dissolved. γ-tert-butyl-L-glutamic acid (2.7 g, 4.04 mmol) was then added. The resulting suspension was allowed to react at 20°C for 2 hours. At the end of the reaction, the solvent was evaporated under reduced pressure, maintaining the jacket temperature at no more than 20°C. 70 mL of n-hexane was added dropwise over approximately 15 minutes, and the mixture was stirred at 20°C under nitrogen for 1 hour until the product was completely crystallized. The resulting pure white solid was filtered on a Buchner funnel and washed with 3×10 mL of n-hexane. It was then dried under vacuum to obtain tert-butyl-NCA monomer (tBu-NCA).

[0051] 5g L-glutamic acid (34mmol) was suspended in 25mL diethylene glycol monomethyl ether solution, and 3mL sulfuric acid was slowly added dropwise at 0°C. After stirring at room temperature overnight, the viscous solution was slowly poured into a mixed solution of triethylamine and isopropanol (volume 1:1). The white solid was collected by two centrifugations (4°C, 6000rpm, 8min) and then dissolved in methanol. After filtration, the filtrate was combined and the solvent was removed under vacuum to obtain a white crude product. The crude product was eluted with a dichloromethane / methanol column chromatography gradient to obtain a white product EG2-Glu. Anhydrous THF (15mL) was added to a mixture of 0.85g EG2-Glu (3.4mmol) and triphosgene (0.39eq) under nitrogen, and the solution was heated to 50°C for 5 hours, after which the solvent was removed under vacuum. The yellow oil was then dissolved in ethyl acetate, loaded onto silica gel for filtration, and the solvent was removed to obtain a light yellow viscous oily product, diethylene glycol-glutamic acid-NCA monomer (EG2-NCA).

[0052] Step 2 Figure 3 ):

[0053] In a nitrogen-filled glove box, difluoroacetic acid (7.48 uL, 0.1188 mmol) was added to a solution of MeO-PEG-NH2 (MW=5000, 600 mg, 0.12 mmol) in ultra-dry DCM (2 mL) to form solution A. 3.57 g, 15.6 mmol of tBu-NCA was added to ultra-dry DCM (8 mL) to form solution B, which was then added to solution A to initiate polymerization. 20 uL of the reaction mixture was taken at each time interval to monitor monomer conversion and the reaction mixture was heated to 100 °C. 1 HNMR peak integration calculation. After 14 hours, the conversion rate reached 86%, and 200uL of solution was used to characterize EG 113 -b-(Glu-tBu) 112Then, the second monomer EG2-NCA (2.59 g, 9.408 mmol) was added to the super dry DCM (5 mL), and the solution was added to the diblock reaction solution and continued to stir at room temperature. 20 uL of the reaction mixture was taken at each time interval to monitor the monomer conversion and 1 Calculation of HNMR peak integration.

[0054] EG 113 -b-(Glu-tBu) 112 -b-(Glu-EG2) 99 The triblock copolymer was purified by precipitation in excess diethyl ether (20 volumes), followed by centrifugation to obtain a white solid, which was then dried overnight in vacuo. The final yield of the triblock copolymer was 68.30%.

[0055] Step 3 Figure 4 ):

[0056] Trifluoroacetic acid (3.87 mL, 0.0521 mmol, 15 equivalents of tert-butyl) was added to EG 113 -b-(Glu-tBu) 112 -b-(Glu-EG2) 99 (1.5 g, 0.0321 mmol) in DCM (4 mL). The reaction mixture was stirred at room temperature. After 48 hours, the reaction was quenched by precipitation with a large amount of cold ether (20 times the volume). The product was purified twice by cold ether precipitation and collected by centrifugation. 113 -b-Glu 112 -b-(Glu-EG2) 99 The triblock co-peptide was obtained as a white solid and dried under vacuum overnight (1.27 g, 97.26%).

[0057] Step 4 Figure 5 ):

[0058] 10mg EG 113 -b-Glu 112 -b-(Glu-EG2) 99 Dissolve in deoxygenated deionized water (3 mL) to form solution C. Dissolve anhydrous FeCl2 (0.8 mg, 5.3 μmol) and FeCl3 (0.3 mg, 2.6 μmol) in deoxygenated deionized water (3 mL) to form solution D. Add solution C and solution D simultaneously into a three-necked flask filled with deoxygenated deionized water (4 mL) and stir under nitrogen at room temperature. Continue the addition for 10 minutes to allow for sufficient intramolecular crosslinking. Stir for another 30 minutes, add 2.5 wt% NH3·H2O, adjust the pH to 11, and allow FeCl2 to form solution D. 2+ / Fe 3+The ions precipitate into Fe₃O₄ and stir at room temperature for 24 hours. The solution is washed twice by centrifugation, and the supernatant is removed. The solution is further dialyzed in a regenerated cellulose dialysis bag (MW cutoff at 1000 Da), then concentrated by centrifugation again and freeze-dried to obtain the Fe-enzyme as a brown powder.

[0059] Step 5 Figure 6 ):

[0060] (Chemical Characterization) X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and high-resolution transmission electron microscopy (HRTEM) analysis of the Fe-enzyme crystal structure revealed an irregular, fragmented structure rather than large aggregates. This structure suggests a high surface-to-volume ratio and potentially a large number of active sites. Infrared spectroscopy (IR) revealed the appearance of new OCO symmetric vibrational peaks after metal coordination. This shift suggests that the carboxyl groups of the second polyglutamic acid segment in the triblock are cross-linked with the metal, a method that is more stable than the commonly used ultrasonic adsorption method. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed the Fe-enzyme morphology as dispersed nanoparticles approximately 10 nm in diameter. Circular dichroism (CD) analysis of the protein's secondary structure revealed that 90% of the triblock structure is α-helical, which is largely converted to β-sheets after metal co-precipitation. This demonstrates that single-chain peptide folded nanozymes can mimic the natural folding of native enzymes and offer a more stable graft structure.

[0061] (Enzymatic Characterization) Electron spin resonance (ESR) was used to detect hydroxyl radicals and superoxide anions. Compared with the blank control group, the hydroxyl radical signal was significantly enhanced, while the superoxide anion signal was weakened, consistent with the action of horseradish peroxidase / CuZn-SOD enzymes, indicating that the Fe-enzyme has dual POD and SOD mimetic activities. Fluorescence intensity measurement of reactive oxygen species (ROS) using a 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe further confirmed the POD mimetic activity of the Fe-enzyme. SOD activity was measured in macrophages (RAW 264.7) after administration of the Fe-enzyme using a SOD kit. The mass percentage of metallic Fe was determined to be less than 5 wt% by inductively coupled plasma mass spectrometry (ICP-MS), indicating that the Fe-enzyme can achieve the above-mentioned mimetic activity at a relatively low metal content.

[0062] (Cell experiment) The triblock polymer and Fe-enzyme had no significant cytotoxicity to normal human lung epithelial cells (BEAS-2B), indicating biological safety. However, the IC 50The value was lower than 2.8 mg / mL, combined with the effect of POD on increasing ROS and leading to tumor cell death, indicating that Fe-enzyme has potential anti-tumor effects.

[0063] (Animal Experiments) The therapeutic effects of Fe-enzyme on oxidative stress and inflammation were investigated in a mouse model of rheumatoid arthritis. Fe-enzyme was injected into the tail vein, and the therapeutic effects of Fe-enzyme on rheumatoid arthritis were analyzed using joint assessment, tissue staining, micro-CT, immunohistochemistry, and oxidative stress markers.

[0064] Example 2

[0065] This embodiment provides a single-chain peptide folding nanozyme, and the preparation method thereof is as follows:

[0066] Steps 1 to 3 in this embodiment refer to Example 1, and in step 4 ( Figure 5 ), 10mg EG 113 -b-Glu 112 -b-(Glu-EG2) 99 Dissolve in deoxygenated deionized water (3 mL) to form solution C. Dissolve MnCl2·4H2O (1.6 mg, 7.9 μmol) in deoxygenated deionized water (3 mL) to form solution D. Add solutions C and D simultaneously dropwise into a three-necked flask filled with deoxygenated deionized water (4 mL) and stir under nitrogen at room temperature. Continue the addition for 10 minutes to allow for sufficient intramolecular crosslinking. Stir for another 30 minutes. Add 2.5 wt% NH3·H2O (200 μL), adjust the pH to 11, and stir at room temperature for 24 hours. Wash the mixture by centrifugation twice, and remove the supernatant. The solution is further dialyzed against a regenerated cellulose dialysis bag (MW cutoff at 1000 Da), then concentrated by centrifugation and freeze-dried to yield the Mn-enzyme.

[0067] The chemical characterization is the same as in Example 1. XPS analysis shows that the Mn element has multiple valence states, including divalent and trivalent, indicating the possibility of dual enzyme activity of POD and SOD. In subsequent activity studies, dual anti-inflammatory and anti-tumor studies can be conducted. (Enzymatic Characterization) The SOD activity value after PANC-1 was administered with Mn-enzyme was detected by SOD kit. The mass percentage of metal Mn was less than 5wt% as determined by inductively coupled plasma mass spectrometry (ICP-MS), indicating that the above enzyme-mimicking activity can be achieved with a lower metal content. (Cellular Experiment) The IC of Mn-enzyme on A549 and PANC-1 50 The value was lower than 1.2 mg / mL, indicating that Mn-enzyme has an anti-tumor cell proliferation effect.

[0068] Example 3

[0069] This embodiment provides a single-chain peptide folding nanozyme, and the preparation method thereof is as follows:

[0070] Steps 1 to 3 in this embodiment refer to Example 1, and in step 4 ( Figure 5 ), 10mg EG 113 -b-Glu 112 -b-(Glu-EG2) 99 Dissolve in deoxygenated deionized water (3 mL) to form solution C. Dissolve anhydrous CoCl2 (1.0 mg, 7.9 μmol) in deoxygenated deionized water (3 mL) to form solution D. Add solutions C and D simultaneously dropwise into a three-necked flask filled with deoxygenated deionized water (4 mL) and stir under nitrogen at room temperature. Continue the addition for 10 minutes to allow for sufficient intramolecular crosslinking. Stir for another 30 minutes. Add 2.5 wt% NH3·H2O (200 μL), adjust the pH to 11, and stir at room temperature for 24 hours. Wash the mixture by centrifugation twice, and remove the supernatant. The solution is further dialyzed in a regenerated cellulose dialysis bag (MW cutoff at 1000 Da), then concentrated by centrifugation again and freeze-dried to obtain the Co-enzyme.

[0071] Chemical characterization was the same as in Example 1. (Enzymatic characterization) The fluorescence intensity of the DCFH-DA probe ROS was used to detect the POD enzyme-mimicking activity of the co-enzyme. The mass percentage of metal Co was determined to be less than 5 wt % by inductively coupled plasma mass spectrometry (ICP-MS), indicating that the co-enzyme can mimic enzyme activity at a relatively low metal content. (Cellular experiments) The IC of the co-enzyme on A549 and PANC-1 was significantly increased. 50 The value was lower than 1.5 mg / mL, indicating that Co-enzyme has an anti-tumor cell proliferation effect.

[0072] Example 4

[0073] This embodiment provides a single-chain peptide folding nanozyme, and the preparation method thereof is as follows:

[0074] Steps 1 to 3 in this embodiment refer to Example 1, and in step 4 ( Figure 5 ), 10mg EG 113 -b-Glu 112 -b-(Glu-EG2) 99Dissolve in deoxygenated deionized water (3 mL) to form Solution C. Dissolve anhydrous CuCl2 (0.5 mg, 4.0 μmol) and ZnCl2 (0.5 mg, 4.0 μmol) in deoxygenated deionized water (3 mL) to form Solution D. Add Solution C and Solution D simultaneously dropwise into a three-necked flask filled with deoxygenated deionized water (4 mL) and stir under nitrogen at room temperature. Continue the addition for 10 minutes to allow for sufficient intramolecular crosslinking. Stir for another 30 minutes. Add 2.5 wt% NH3·H2O (200 μL), adjust the pH to 11, and stir at room temperature for 24 hours. Wash the mixture by centrifugation twice, and remove the supernatant. The solution is further dialyzed against a regenerated cellulose dialysis bag (MW cutoff at 1000 Da), then concentrated by centrifugation and freeze-dried to yield the CuZn-enzyme.

[0075] The chemical characterization content is the same as that of Example 1. The XPS 2p Cu / 2p Zn spectrum of CuZn-enzyme is compared with the 2p Cu of Cu-enzyme and the 2p Zn of Zn-enzyme, and it is found that copper / zinc are well intercalated in the block polymer. (Enzymatic characterization) Electron spin resonance (ESR) is used to detect superoxide anions. Compared with the blank control group, the superoxide anion signal is weakened, which is consistent with the effect of CuZn-SOD enzyme, indicating that CuZn-enzyme has SOD mimetic enzyme activity. The mass percentage of metal Cu / Zn is less than 2wt% determined by inductively coupled plasma mass spectrometry (ICP-MS), indicating that it can play a mimetic enzyme activity role with a lower metal content. (Cell experiment) The SOD kit detects the SOD activity value after PANC-1 is administered with CuZn-enzyme. The IC values ​​of CuZn-enzyme for A549 and PANC-1 50 The value was lower than 0.4 mg / mL, indicating that CuZn-enzyme has an anti-tumor cell proliferation effect.

[0076] Comparative Example 1

[0077] This comparative example adopts the enzymatic characterization method in the fifth step of Example 1 to measure the peroxidase activity of the natural enzyme horseradish peroxidase (HRP) / Fe3O4, which first proposed the concept of nanozyme.

[0078] The ESR spectra of the free generation of hydroxyl groups in Example 1 and Comparative Example 1 are compared as follows: Figure 7 The results show that the metal solid content of Fe3O4 is 72wt%, while the metal solid content of Fe-enzyme in the present invention is less than 5wt%. The metal ratio of Fe-enzyme in the present invention is lower, and the enzyme activity is closer to that of natural enzyme ( Figure 7 ).

[0079] Comparative Example 2

[0080] In this comparative example, two Fe3O4 nanoparticles with diameters of 10 nm and 20 nm were selected for comparison with the Fe-enzyme in Example 1.

[0081] The results showed that for A549 cell line, although the intracellular iron level of Fe-enzyme (78.44ng / 10 4 cells) Fe3O4 nanoparticles below 10nm and 20nm (147.65 and 186.02ng / 10 4 cells), but we must consider the net metal content. For Fe-enzyme, the net iron content is only 2.91 wt%, while for commercial Fe₃O₄, the net iron content is 72.4 wt%. Therefore, the Fe-enzyme nanoparticles absorbed 12.8 times more than 10 nm Fe₃O₄ and 10.8 times more than 20 nm Fe₃O₄. For PANC-1 cells, the Fe-enzyme nanoparticles were taken up 31.5 times more than 10 nm Fe₃O₄ and 3.9 times more than 20 nm Fe₃O₄. This indicates that the PEG-polypeptide block copolymer significantly enhances ferritin cellular uptake compared to unbound Fe₃O₄ nanoparticles.

[0082] Comparative Example 3

[0083] In this comparative example, steps 1 to 3 refer to Example 2, and in step 4 ( Figure 5 ):

[0084] (1) 10mg EG 113 -b-Glu 112 -b-(Glu-EG2) 99 Dissolve in deoxygenated deionized water (3 mL) to form solution C. Dissolve MnCl2·4H2O (5.3 mg, 26.3 μmol) in deoxygenated deionized water (3 mL) to form solution D. Add solutions C and D simultaneously dropwise into a three-necked flask filled with deoxygenated deionized water (4 mL) and stir under nitrogen at room temperature. Continue the addition for 10 minutes to allow for sufficient intramolecular crosslinking. Stir for another 30 minutes. Add 2.5 wt% NH3·H2O (200 μL), adjust the pH to 11, and stir at room temperature for 24 hours. Wash the mixture by centrifugation twice, and remove the supernatant. The solution is further dialyzed against a regenerated cellulose dialysis bag (MW cutoff at 1000 Da), then concentrated by centrifugation and freeze-dried to yield the Mn-enzyme.

[0085] (2) 10mg EG 113 -b-Glu 112 -b-(Glu-EG2) 99Dissolve in deoxygenated Dl water (3 mL) to form solution C. Dissolve anhydrous CoCl2(3.3 mg, 26.1 μmol) in deoxygenated Dl water (3 mL) to form solution D. Solution C and solution D were simultaneously added dropwise into a three-neck flask filled with deoxygenated Dl water (4 mL) under nitrogen stirring at room temperature. The dropwise addition process lasted 10 min, and the intramolecular cross-linking was allowed to proceed for another 30 min. 2.5 wt%, 200 μL of NH3H2O was added to adjust the pH to 11, and the solution was stirred at room temperature for 24 h. The solution was washed twice by centrifugation, and the supernatant was removed. The solution was further dialyzed in a regenerated cellulose dialysis bag (M.W. cut off at 1000 Da), and then concentrated again by centrifugation and freeze-dried to obtain Co-enzyme.

[0086] (3) Dissolve 10 mg of EG 113 -b-Glu 112 -b-(Glu-EG2) 99 Dissolve in deoxygenated Dl water (3 mL) to form solution C. Dissolve anhydrous CoCl2(3.3 mg, 26.1 μmol) in deoxygenated Dl water (3 mL) to form solution D. Solution C and solution D were simultaneously added dropwise into a three-neck flask filled with deoxygenated Dl water (4 mL) under nitrogen stirring at room temperature. The dropwise addition process lasted 10 min, and the intramolecular cross-linking was allowed to proceed for another 30 min. 2.5 wt%, 200 μL of NH3H2O was added to adjust the pH to 11, and the solution was stirred at room temperature for 24 h. The solution was washed twice by centrifugation, and the supernatant was removed. The solution was further dialyzed in a regenerated cellulose dialysis bag (M.W. cut off at 1000 Da), and then concentrated again by centrifugation and freeze-dried to obtain Co-enzyme.

[0087] IC values of the three kinds of nanoparticles on BEAS-2B cells after 24 h of administration were detected, and the detection results are shown in Table 3. 50 values of the three kinds of nanoparticles on BEAS-2B cells after 24 h of administration were detected, and the detection results are shown in Table 3.

[0088] Table 3 IC values of the three kinds of nanoparticles on BEAS-2B cells after 24 h of administration were detected, and the detection results are shown in Table 3. 50 values of the three kinds of nanoparticles on BEAS-2B cells after 24 h of administration were detected, and the detection results are shown in Table 3.

[0089]

[0090] In the present comparative example, the molar ratio of metal to carboxyl group of the polymer was increased from 1:3.3 in Comparative Example 2 to 1:1. Table 3 shows that the Mn-enzyme, Co-enzyme and CuZn-enzyme synthesized in the present comparative example still have enzyme-like activity, but their cytotoxicity increases, especially to normal cells BEAS-2B.

[0091] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing a single-chain peptide folding nanozyme, characterized in that: The following steps are involved: (a) synthesizing an NCA (N-carboxylic anhydride) monomer having a side chain with a group that can be hydrolyzed to a carboxyl group and an amino acid NCA (N-carboxylic anhydride) monomer having a hydrophilic group on the side chain; (b) preparing a single-chain triblock polymer comprising an initiator segment, a middle segment, and a terminal segment by sequential ring-opening polymerization; wherein the middle segment is a group-NCA polymer that can be hydrolyzed to a carboxyl group, and the terminal segment is a hydrophilic amino acid-NCA polymer; (c) hydrolyzing the groups in the middle segment that can be hydrolyzed into carboxyl groups into carboxyl groups to form a single-chain polyamino acid polymer having a polyamino acid chain as the middle segment; (d) The carboxyl group is coordinated and complexed with a transition metal ion to fold the middle segment of the polymer, and then the complexed metal ions are converted into corresponding metal oxides and hydroxides by coprecipitation. The resulting enzyme-mimicking nanoparticles are the single-chain peptide folded nanozyme.

2. The preparation method according to claim 1, characterized in that In the step (a), the side chain has a group that can be hydrolyzed to form a carboxyl group - in the NCA monomer, the group that can be hydrolyzed to form a carboxyl group is any one or more of a methyl ester group, an ethyl ester group, a tert-butyl ester group, an n-butyl ester group, a benzyl group, a substituted benzyl group, a diphenylmethyl ester group, an amide group, a hydrazide group, a potassium salt, a sodium salt and a triethylamine salt; the NCA parent amino acid is any one or more of aspartic acid, glutamic acid, lysine and arginine.

3. The preparation method according to claim 2, characterized in that In the amino acid-NCA monomer having a hydrophilic group on the side chain in step (a), the hydrophilic group includes an ethylene glycol oligomer, and the ethylene glycol oligomer is any one or more of monoethylene glycol, diethylene glycol and triethylene glycol; the NCA parent amino acid is any one or more of aspartic acid, glutamic acid, lysine and arginine.

4. The preparation method according to claim 3, characterized in that The initiating segment in step (b) is a substance in which one segment is amino and the other segment is methoxy, which can be any one or more of polyethylene, polystyrene, methoxy-polyethylene glycol-amino, polymethyl methacrylate, polyacetic acid lactone, polylactic acid and polyethylene glycol monomethyl ether.

5. The preparation method according to claim 4, characterized in that The hydrolysis reagent in step (c) is any one or more of acetic acid, hydrochloric acid, hydrogen bromide, potassium hydroxide, sodium borohydride, trifluoroacetic acid and boron trifluoride diethyl ether; the number average molecular weight of the single-chain polyamino acid is 100 to 3,000,000.

6. The preparation method according to claim 5, characterized in that The metal ion used for coordination and cross-linking in step (d) is Fe 2+ 、Fe 3+ 、Cu 2+ 、Zn 2+ 、Mn 2+ 、Mn 3+ 、Mn 4+ 、Co 2+ 、Co 3+ Cr 2+ Cr 3+ , Pt 2+ , Pt 4+ 、Au + 、Au 3+ 、Ag + , Ca 2+ any one or more of; the precipitant used for co-precipitation includes alkali, hydrogen sulfide and / or sulfide salt, wherein the alkali is any one or more of NH3·H2O, NaOH, KOH, Ca(OH)2, CsOH, and the sulfide salt is any one or more of Na2S, K2S, Cs2S.

7. The preparation method according to claim 6, characterized in that The solvent used for the biomimetic folding of polyamino acids in step (d) should be a solvent that can dissolve the three segments, specifically any one or more of dimethyl sulfoxide (DMSO), dichloromethane (DCM), water (H2O), 3,3'-dichlorobenzidine (DCB), dimethylacetamide (DMA), ethylene glycol, aniline, acetic acid, nitromethane, pyridine, dioxane and dimethyl phthalate.

8. A single-chain peptide folded nanozyme prepared using the preparation method according to any one of claims 1 to 7.

9. Use of the single-chain peptide folding nanozyme according to claim 8 in the preparation of anti-tumor drugs and / or anti-inflammatory drugs, characterized in that: Among them, single-chain peptide folding nanozymes with peroxidase (POD) and / or oxidase (OXD) activities are used to prepare anti-tumor drugs; single-chain peptide folding nanozymes with superoxide dismutase (SOD) and / or catalase (CAT) activities are used to prepare anti-inflammatory drugs.

10. A drug, characterized in that It contains the single-chain peptide folding nanozyme according to claim 8.

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