Self-assembled polypeptide nano-enzyme and application thereof

Abstract

The invention discloses a self-assembled polypeptide nano-enzyme and application thereof, the self-assembled polypeptide nano-enzyme comprises a polypeptide scaffold, a polypeptide sequence with targeting and nano-enzyme affinity, a main body with amino acid having a characteristic sequence derived from natural enzyme and a functional motif containing hydrophobic peptide, the polypeptide nanoparticles are self-assembled by utilizing the hydrophobic interaction of amino acids; the nano enzyme is in coordination binding with the central coordination amino acid; and the targeting peptide is positioned at the N end of the polypeptide sequence and is displayed on the outer layer surface of the self-assembled polypeptide nanoparticle. According to the invention, the high-activity nano-enzyme is biomimetically designed by simulating a natural enzyme metal ion active site and a spatial structure of a catalytic microenvironment. The novel polypeptide nano-enzyme with targeting property and biosafety is constructed by taking polypeptide as a synthesis template. The self-assembled polypeptide nano-enzyme can be administrated through aerosol inhalation, targeted positioning of the nano-enzyme on a focus is guided, ROS of focus tissue is regulated and controlled, and occurrence and development of diseases are inhibited.

Description

Technical Field

[0001] The present invention belongs to the cross-field of nanobiology, nanoenzymology, biomimetic synthesis and biomedicine, and specifically relates to a self-assembling polypeptide nanozyme and application thereof. Background Art

[0002] Nanozymes are a type of nanomaterials with enzymatic properties. They have enzymatic reaction kinetics similar to those of natural enzymes and can be used as substitutes for enzymes for human health. They have the advantages of high stability, easy preparation, low cost, easy surface modification, adjustable catalytic activity, and easy large-scale production. They overcome the limitations of natural enzymes and have the designability, versatility and manipulability of nanomaterials. They show great application potential in industry, medicine, biology and other fields, and were rated as one of the top ten emerging chemical technologies by the International Union of Pure and Applied Chemistry (IUPAC) in 2022. Nanozyme-mediated catalytic therapy has broad application prospects in the treatment of diseases related to oxidative stress. For example, Fe in dendritic silicon nanoparticles 3 O 4 Nanozymes can effectively consume glucose in tumor cells through glucose oxidase (GOx)-like activity, generate reactive oxygen species (ROS) and lead to tumor cell apoptosis. Mn nanozymes with multi-enzyme activities similar to SOD, CAT, and glutathione peroxidase (GPx) can effectively consume glucose in tumor cells through glucose oxidase (GOx)-like activity, generate reactive oxygen species (ROS) and lead to tumor cell apoptosis. 3 O 4 Nanozymes for Parkinson's disease neuroprotection, MoS with SOD-like and CAT-like activities 2 Nanozymes are used to treat osteoarthritis, etc.

[0003] Nanozymes have shown great potential as an emerging catalytic therapeutic strategy, but there are still some bottlenecks that limit the further development of nanozymes. The main challenge of nanozymes in biomedical applications is how to give them higher catalytic activity and lesion targeting ability. Compared with natural enzymes and organic catalysts, the catalytic activity and efficiency of nanozymes are generally relatively low, which limits their application in disease treatment. The uneven atomic composition and complex crystal surface structure of nanozymes make it difficult to identify the actual catalytic active center and reveal the enzyme-like activity mechanism. Simulating the structure and enzymatic microenvironment of the active center of natural enzymes to biomimetic design of nanozymes has been proven to be a preferred strategy to improve the activity of nanozymes. Unlike traditional enzymes, current nanozymes are mostly inorganic nanoparticles, which may damage cells through irreversible interactions with intracellular components after entering cells. In particular, metal-based nanozymes are prone to systemic toxicity after administration due to degradation and release of metal ions, and appropriate targeted functional modifications (functional groups, targeting peptides, etc.) are required to improve the enrichment of nanozymes at the lesion site.

[0004] Nanozymes reported in current studies usually lack selectivity and specificity, have insufficient biosafety, and generally have limited catalytic activity, making them unable to achieve effective clinical applications. Therefore, biomimetic design of biosafe and highly active nanozymes will provide a new strategy for catalytic therapy. The metal active site and the surrounding protein scaffold jointly determine the catalytic activity of natural enzymes. The catalytic function of enzymes depends largely on the chemical structure of the active site, which is composed of amino acid residues in the primary sequence arranged into a three-dimensional groove. In these active sites, functional groups cooperate with cofactors (organic or ionic) to specifically bind substrates and catalyze their conversion. Reconstructing the active site of enzymes in artificial systems to create stable biocatalysts remains a major challenge. Therefore, the co-simulation of the spatial structure of the metal active site and the protein scaffold microenvironment is expected to be an effective way to biomimetic design of highly active nanozymes.

[0005] In summary, how to further achieve the high efficiency and safety of catalytic reactions is a bottleneck problem that nanozymes urgently need to break through.

[0006] Natural biomacromolecules with unique spatial structures can be used as important tools for the development of nanozymes. As a type of biogenic material, peptides have received extensive attention in the field of disease treatment and diagnosis due to their good biocompatibility and convenience of biofunctionalization. Multifunctional self-assembling peptide nanocarriers can be constructed through genetic engineering technology. Peptide nanoparticles modified with targeting peptides and functional peptides can accurately deliver drugs to lesions, achieving environmentally responsive targeted aggregation and controllable drug release. In addition, the precisely controllable amino acid sequence of peptides can not only form a variety of spatial configurations as needed, but also can be efficiently combined with other bioactive moieties to construct responsive multifunctional nanosystems. Studies have shown that amino acids can form active sites after affinity with specific metal ions, exhibiting different enzyme-like catalytic activities.

[0007] At present, most of the biomimetic nanozymes are nanomaterials such as metal organic frameworks synthesized by chemical methods, which have limited specificity and cause potential toxicity (CN 114768872A), while the reported polypeptide nanozymes are usually small peptide sequences based on simple amino acids, which are difficult to form three-dimensional spatial structures, greatly affecting the improvement of catalytic activity (CN 116970584A, CN114457050B), making it difficult to achieve industrial transformation and unable to meet clinical needs. Therefore, rationally designing self-assembling polypeptides to form ordered nanostructures can be used as biomineralization templates to synthesize nanozymes with high catalytic activity and biosafety, showing great application potential in the field of biomedical catalytic therapy.

[0008] It is worth noting that previous nanozyme therapies were mainly administered by intravenous injection or in situ injection. For lung diseases, atomization inhalation is a more convenient local administration method. The drug solution or powder can be atomized into ultrafine aerosol particles through a compressed atomizer generator suitable for lower respiratory tract diseases. It is inhaled and deposited in the lower respiratory tract and alveoli, thereby improving symptoms and treating lung diseases. Atomization administration is conducive to the rapid diffusion of drugs, directly reaching lung lesions and contacting diseased tissues. It has the advantages of being non-invasive, fast-acting, and having few systemic side effects. In the field of nanodrug delivery, many types of nanoparticles have been reported to be prepared by specific devices to form aerosols for administration. However, there are currently no reports on the use of nanoenzymes in the form of atomization inhalation to treat lung diseases. Summary of the invention

[0009] In response to the problems of low efficiency and toxicity of the above-mentioned nanozyme catalytic reaction, the present invention provides a self-assembling polypeptide nanozyme and its application. The polypeptide nanozyme simulates the spatial structure of the active center of the natural enzyme and the microenvironment of the catalytic center to obtain efficient enzyme-like catalytic activity. The targeting peptide is displayed on the surface of the nanozyme to guide the aggregation of the nanozyme in the lesion by utilizing the affinity between the targeting peptide and its receptor. It can not only target the treatment of diseases, but also provide a precise, efficient and biosafe polypeptide nanozyme catalytic treatment plan for other oxidative stress-related diseases.

[0010] In order to achieve the above object, the present invention provides a self-assembling polypeptide nanozyme, which comprises: Peptide scaffolds with targeting and nanozyme affinity peptide sequences, including: The main body, whose amino acids have characteristic sequences derived from natural enzymes, is composed of amino acids coordinating the active center of natural enzymes and amino acids participating in the formation of α-helical structures in the conserved sequences of natural enzymes. Contains a functional motif of hydrophobic peptides, which self-assemble into peptide nanoparticles using the hydrophobic interactions of amino acids; A nanozyme, coordinated and bound to the central coordinating amino acid; The targeting peptide is located at the N-terminus of the polypeptide sequence and is displayed on the outer surface of the self-assembled polypeptide nanoparticles, such as the receptor angiotensin converting enzyme 2 targeting peptide STSQKSIVAYTM, the tumor targeting peptide GRGDS, the tumor targeting peptide QHWSYGLRPG, the fibrin targeting peptide CREKA, the brain capillary endothelial cell targeting peptide HAIYPRH, the macrophage targeting peptide YEQDPWGVKWWY, the nerve cell targeting peptide CLEVSRKNC, the cardiomyocyte targeting peptide CSTSMLKAC, etc.

[0011] The present invention uses genetic engineering technology to bionic design and screen a self-assembling polypeptide nanozyme based on the structure-activity relationship of natural enzymes. The amino acids in the main part of the polypeptide are derived from the characteristic sequence of natural enzymes, and the polypeptide self-assembly is used as a scaffold to synthesize the nanozyme. On the one hand, by simulating the spatial structure of the active center of the natural enzyme and the microenvironment of the catalytic center, efficient enzyme-like catalytic activity is obtained. On the other hand, by drawing on the affinity of the targeting peptide with the cell surface receptor, the targeting peptide for the target tissue is displayed on the surface of the nanozyme to guide the aggregation of the nanozyme in the target lesion.

[0012] Preferably, the motif of the main body comprises 5-50 amino acids, which is an amino acid sequence capable of forming a tertiary structure.

[0013] Further preferably, the amino acid sequence of the motif of the subject is QDWEH, QDEWEH, KFHGGGDKNHQDHHDKWEH, LKHLDGLRLHGKDNQDHHEK, KFEDGEHYHRHQDSWEH, KFEDGEAYHRHQDSWEH, KFEDGEHYARHQDSWEH, KFEDGEHYHRAQDSWEH, KFEDGEHYHRHQDSWEA, KFEDGEHYHRHQASWEH, KFEDGEHAHRHQDSWEH, KFEDGEHYHRHADSWEH, KFEDGEHYHRHQDSAEH or KAEDGEHYHRHQDSWEH.

[0014] Further preferably, the motif of the subject comprises 5-17 amino acids.

[0015] Preferably, the hydrophobic peptide-containing functional motif comprises 5-50 amino acids.

[0016] Further preferably, the hydrophobic amino acid containing the functional motif of the hydrophobic peptide is one or any combination of A, F, I, L, M, P, V, W, Y, or a hydrophobic polypeptide with biological function.

[0017] Furthermore, the functional motif containing the hydrophobic peptide is a combination sequence of AYYLLY, WLFLL, AYYLQYAYYL, PALWLFLVLL or AWYFWAFLWL.

[0018] Preferably, the nanozyme is a complex formed by a metal ion and an amino acid and / or a cofactor, a metal, or a metal oxide.

[0019] Furthermore, the cofactor is porphyrin, macrocyclic polyamines such as deferoxamine (DFOB), 4-chloro-2,6-dipropionylpyridine, amines such as nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), etc., preferably porphyrin. These substances can chelate with metal ions to form complexes, and provide nitrogen in the complexes to form a coordination structure of metal and nitrogen.

[0020] The above design methods of self-assembling peptide nanozymes include: 1) By analyzing the structure of natural enzymes, simulating the spatial structure of the active site of natural enzymes and the amino acid composition of the catalytic center microenvironment, rationally designing and synthesizing a series of polypeptide sequences, self-assembling polypeptide nanoparticles in aqueous solution, and using them as a scaffold to complex metal ions with porphyrin and other complexes, a series of self-assembling polypeptide nanozymes were prepared. The enzyme-like catalytic activity was detected, and the self-assembling polypeptide nanozyme with the highest catalytic activity was screened out. The structural characteristics of the self-assembling polypeptide nanozyme were analyzed by characterizing its morphology, size, metal ion valence and ratio, metal ion coordination with porphyrin and amino acids, secondary structure and other properties to ensure the stability of the properties before and after atomization.

[0021] 2) The coordination structure and the molecular mechanism and dynamics of enzyme-like catalytic activity were studied through theoretical calculations, which are consistent with the characteristics of the active center of natural enzymes.

[0022] The above-mentioned peptide nanozyme realizes a new therapeutic strategy that combines cascade targeting, lysosomal escape, and cascade controllable catalytic activity. Through the synergistic effect of peptides and nanozymes, targeted aggregation of nanozymes is achieved, enzyme catalytic activity is improved, and toxic side effects are reduced, achieving better catalytic therapeutic effects. Specifically: 1) The present invention explores the ability of self-assembling peptide nanozymes to target and bind to cell or tissue surface receptors in vitro. The fluorescent probe FITC-labeled nanozymes are co-localized with lysosomes and mitochondria to verify the distribution and targeted aggregation of peptide nanozymes in cells. The ability of self-assembling peptide nanozymes to regulate intracellular ROS is tested in vitro to characterize their cytotoxicity and effects on cell viability.

[0023] 2) In vivo characterization of the biodistribution and clearance of self-assembling peptide nanozymes, application dosage, tissue targeting, therapeutic effects on disease models, and biosafety. Systematically study the regulation of self-assembling peptide nanozymes on the lesion microenvironment and clarify the mechanism of action.

[0024] The second aspect of the present invention provides the use of the above-mentioned self-assembling polypeptide nanozyme in the preparation of a nebulized inhalation therapeutic drug for lung diseases. The above-mentioned self-assembling polypeptide nanozyme system has unchanged structure and properties after atomization, and can be used for a nebulized inhalation administration method. Through aerosol inhalation, targeted regulation of free radicals in lung disease tissues through pulmonary administration is achieved, thereby achieving precise targeted treatment of respiratory diseases. The use of this self-assembling polypeptide nanozyme with high enzyme activity and targeting for the nebulized inhalation treatment of lung diseases not only provides a new administration method for nanozymes, but also provides new strategies and new ideas for the clinical treatment of patients with respiratory diseases.

[0025] Through the above technical solution, the present invention achieves the following beneficial effects: The present invention designs a highly active nanozyme by simulating the spatial structure of the metal ion active site and the catalytic microenvironment of the natural enzyme. A novel polypeptide nanozyme with targeting and biosafety is constructed using a polypeptide as a synthetic template. This self-assembling polypeptide nanozyme can be administered by aerosol inhalation, guiding the targeted positioning of the nanozyme in the lesion, regulating the ROS of the lesion tissue, and inhibiting the occurrence and development of the disease. This novel bionic self-assembling polypeptide nanozyme with high enzyme activity and targeting is used for the aerosol inhalation treatment of lung diseases, which can provide new strategies and new ideas for the clinical treatment of lung disease patients. Compared with the small molecule drugs currently used in clinical practice, this inhaled self-assembling polypeptide nanozyme is a targeted treatment strategy that effectively reduces toxic side effects and ensures bioavailability and safety. The biomimetic self-assembling polypeptide nanozyme provides more efficient catalytic activity and stability, and is a new catalytic treatment solution with high efficiency and low toxicity. In addition, this self-assembling polypeptide nanozyme platform established by biomimetic design based on structure-activity relationship can be used for a variety of specific receptor targeted modifications, combined with the simulation of a variety of natural enzyme active centers and catalytic microenvironments, for a wide range of disease treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic representation of the SOD-like and CAT enzyme activities (n=3) of the self-assembling polypeptide nanozyme in Example 1 of the present invention; Figure 2 is a transmission electron microscope (TEM) image of the self-assembling polypeptide nanozyme in Example 1 of the present invention; Figure 3 This is the circular dichroism (CD) spectrum of the self-assembled polypeptide nanozyme before and after atomization in Example 1 of the present invention; Figure 4 is the X-ray diffraction (XRD) of the self-assembling polypeptide nanozyme of Example 1 of the present invention; Figure 5 is the ultraviolet-visible (UV-vis) spectrum of the self-assembling polypeptide nanozyme of Example 1 of the present invention; Figure 6is the X-ray photoelectron spectroscopy (XPS) spectrum of the self-assembling polypeptide nanozyme of Example 1 of the present invention; Figure 7 is the Fourier transform infrared spectrum (FTIR) of the self-assembling polypeptide nanozyme of Example 1 of the present invention; Figure 8 This is the Raman spectrum of the self-assembling polypeptide nanozyme of Example 1 of the present invention; Fig. 9 This is the liquid nuclear magnetic resonance spectrum of the self-assembling polypeptide nanozyme of Example 1 of the present invention; Fig.10 It is PepNzyme in Example 2 of the present invention SOD-CAT Verification of cascade enzyme activity at the cell level, where a is the verification of SOD activity at the cell level, b is the verification of CAT activity at the cell level, and c is the verification of H in hACE2-293T cells. 2 O 2 d is the clearance of ROS in hACE2-293T cells (n=3); Fig.11 is a schematic diagram of the cascade targeting function at the cellular level in Example 3 of the present invention; Fig.12 is the CLSM cell-targeted binding analysis in Example 3 of the present invention (n=3), the scale bar represents 50 μm; Fig.13 is the lysosomal escape and mitochondrial aggregation in Example 3 of the present invention (n=3), the scale bar represents 10 μm; Fig.14 is the mitochondrial regeneration in Example 3 of the present invention (n=3), the scale bar represents 2 µm; Fig.15 is the CLSM analysis of ACE2 targeted co-localization in lung tissue in Example 3 of the present invention (n=3), and the scale bar represents 200 μm; Fig.16 The IVIS in Example 3 of the present invention is used to image the lungs and other organs including the heart, liver, spleen and kidneys; Fig.17 is the quantitative analysis of the Cy5 fluorescence signal in the organs collected from PepNzyme-operated mice in Example 3 of the present invention (n=3); Fig.18 is the tissue-cleared Mn content measured by ICP-MS in Example 3 of the present invention (n=3); Fig.19 This is the evaluation of apoptosis in lung tissue by TUNEL in Example 3 of the present invention, and the scale bar represents 200 μm; Fig. 20It is a schematic diagram of the experimental process in Example 4 of the present invention; Fig.21 are in vitro images of lung tissue 5 days and 30 days after treatment with PepNzyme in Example 4 of the present invention; Fig. 22 is the concentration of CRP in BALF in Example 4 of the present invention; Fig.23 This is HE staining of lung tissue in Example 4 of the present invention, with a scale of 50 micrometers; Fig.24 is the inflammatory response score in Example 4 of the present invention; Fig.25 It is the Masson staining of lung tissue (muscle fibers and erythrocytes (red), collagen (blue), and nuclei (black-purple)) in Example 4 of the present invention, with a scale of 200 μm; Fig.26 The Ashcroft scoring method used in Example 4 of the present invention is a quantitative analysis of mouse pulmonary fibrosis by a blind method; Fig. 27 This is the hematoxylin-eosin staining condition in Example 4 of the present invention; Fig.28 This is the quantitative analysis of mouse lung tissue fibrosis in Example 4 of the present invention, with a scale of 50 micrometers; Fig.29 is a representative immunofluorescence image of COL1A1 and α-SMA in mouse lung tissue sections in Example 4 of the present invention, with a scale of 100 μm; Fig.30 It is the lung permeability study conducted by Evans blue in Example 4 of the present invention; Fig.31 This is the lung wet / dry weight analysis in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0028] Example 1 Rational design, preparation and screening of self-assembling polypeptide nanozymes method 1. Rational design and synthesis of self-assembling peptide scaffold sequences By analyzing and simulating the spatial structure of the active sites of natural MnSOD and MnCAT enzymes and the amino acid composition of the catalytic center microenvironment, a series of nanozyme affinity peptide sequences were rationally designed and synthesized by Nanjing Peptide Valley Biological Company, including a series of peptide sequences (SEQ ID No. 1-5) containing different proportions of α-helical amino acids (S-1 33.33%, S-2 16.67%, L-1 69.05%, L-2 9.52%, L-3 32.5%). The amino acid composition of these peptides is derived from the characteristic sequences of MnSOD and MnCAT, including the residues responsible for coordinating with the Mn ion in the active center of the native enzyme (H26, H74, H163, D159 in MnSOD and E36, E70, E155, H73, H188 in MnCAT), specific conserved amino acid sequences involved in the formation of the α-helical structure in MnSOD (K64, G68, Q141, D142; KFEDGEHY--EHAWY) and HXXE contained in the α-helical structure in MnCAT, as well as amino acids that have been reported to inhibit the activity of MnSOD (Y34, Q143, W161, E162) or MnCAT (Y42, F2) after mutation. The peptide sequence containing 32.5% α-helical amino acid residues includes a rationally designed sequence (L-3) and a series of mutant sequences, each of which mutates one of the amino acids (in the L-3 sequence, 9 amino acids H1, H2, H3, H4, D159, Y34, Q143, W161, E162, and F2 are replaced by A (alanine)) (SEQ ID No.6-14). The hydrophobic residues in the peptide mimic the hydrophobic environment of the active sites of natural MnSOD and MnCAT enzymes, and promote their self-assembly into nanoparticles through hydrophobic interactions. The targeting peptide SP10 (STSQKSIVAYTM) is designed at the N-terminus of the peptide sequence. The targeting peptide comes from the RBD region of the new coronavirus and can be displayed on the outer surface of the self-assembled peptide nanoparticles. It binds to the receptor ACE2 highly expressed in lung tissue through inhalation, thereby guiding the peptide nanozyme to target the lung lesions.

[0029] SEQ ID No.1 (S-1): STSQKSIVAYTM-P-QDWEHAYYLLY SEQ ID No.2 (S-2): STSQKSIVAYTM-P-QDEWEHWLFLL SEQ ID No.3 (L-1): STSQKSIVAYTM-G-KFHGGGDKNHQDHHDKWEHAYYLQYAYYL SEQ ID No.4 (L-2): STSQKSIVAYTM-P-LKHLDGLRLHGKDNQDHHEKPALWLFLVLL SEQ ID No.5 (L-3): STSQKSIVAYTM-G-KFEDGEHYHRHQDSWEHAWYFWAFLWL SEQ ID No.6 (H1A): STSQKSIVAYTM-G-KFEDGEAYHRHQDSWEHAWYFWAFLWL SEQ ID No.7 (H2A): STSQKSIVAYTM-G-KFEDGEHYARHQDSWEHAWYFWAFLWL SEQ ID No.8 (H3A): STSQKSIVAYTM-G-KFEDGEHYHRAQDSWEHAWYFWAFLWL SEQ ID No.9 (H4A): STSQKSIVAYTM-G-KFEDGEHYHRHQDSWEAAWYFWAFLWL SEQ ID No.10 (D159A): STSQKSIVAYTM-G-KFEDGEHYHRHQASWEHAWYFWAFLWL SEQ ID No.11 (Y34A): STSQKSIVAYTM-G-KFEDGEHAHRHQDSWEHAWYFWAFLWL SEQ ID No.12 (Q143A): STSQKSIVAYTM-G-KFEDGEHYHRHADSWEHAWYFWAFLWL SEQ ID No.13 (E162A): STSQKSIVAYTM-G-KFEDGEHYHRHQDSAEHAWYFWAFLWL SEQ ID No.14 (F2A): STSQKSIVAYTM-G-KAEDGEHYHRHQDSWEHAWYFWAFLWL 2. Preparation of Mn-based self-assembled polypeptide antioxidant nanozyme Mn simulating natural Mn-SOD3+ Active center, with self-assembled peptide nanoparticles as scaffold and MnCl 2 Mn was synthesized from 4-phenylporphyrin and 4-phenylporphyrin in a weakly alkaline AMPSO buffer at pH 8. 3+ Porphyrin is a biosafe complexing agent that can complex Mn 2+ And promote its oxidation to generate corresponding Mn 3+ Coordination compounds, porphyrin as a ligand, on the one hand, will promote Mn 3+ On the other hand, it can quickly stabilize the product Mn 3+ At the same time, the amino acids on the polypeptide scaffold that are affinity with Mn metal ions can react with Mn 3+ , porphyrin coordination, the imidazole ring of the amino acid and the porphyrin ring of 4-phenylporphyrin bind to Mn 3+ An active center structure similar to the natural enzyme Mn-SOD is formed, thereby preparing a self-assembling polypeptide Mn-based nanozyme with multiple active centers.

[0030] The specific steps are as follows: 1 mg of peptide was dissolved in 1.7 ml of water, and then 0.5 M NaOH solution was added dropwise to adjust the pH value of the solution to 8. At room temperature, 0.1 ml (50 mM) MnCl was added dropwise within 20 seconds under constant stirring. 2 Then add 0.2 ml (50 mM) 4-phenylporphyrin and react for 5 hours. Keep the lid open during the reaction to ensure adequate air contact.

[0031] 3. Optimization screening and structural property characterization of highly active self-assembling peptide nanozymes The superoxide anion scavenging activity of a series of self-assembling peptide nanozymes was tested using a SOD activity assay kit. To evaluate CAT activity, 300 µM H 2 O 2 Observe the bubbles produced. Add 80 mM H 2 O 2 and 0.02 mg / mL PepNzymes, and then the H 2 O 2 The characteristic absorbance (wavelength 240 nm) was used to detect H 2 O 2 The elimination of oxidative stress was detected by PCR. The self-assembling peptide antioxidant nanozyme with the highest catalytic activity was selected for subsequent research. After screening, the peptide nanozyme PepNzyme based on the peptide sequence L-3 was selected. SOD-CAT Shows extremely strong SOD and CAT catalytic activity.

[0032] Transmission electron microscopy (TEM) was used to characterize the morphology and size of the self-assembled peptide antioxidant nanozymes, and ultraviolet spectrophotometer (UV-vis), X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma mass spectrometry (ICP-MS) were used to characterize the valence and ratio of Mn ions. Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray diffraction (XRD), liquid nuclear magnetic resonance and circular dichroism (CD) were used to investigate the coordination of Mn ions with porphyrin and amino acids, as well as the PepNzyme SOD-CAT The structural features were characterized.

[0033] The polypeptide nanozyme solution was injected into a compression nebulizer, and the drug was dispersed into fine droplets by a jet atomization method through a high-pressure oxygen flow. The polypeptide nanozyme solution was sprayed into a collection tube and then re-condensed. The condensate was collected for the above characterization, and the structural properties and catalytic activity of the polypeptide nanozyme before and after atomization were compared.

[0034] result A series of peptide sequences containing targeting motifs and Mn ion affinity were designed to promote their self-assembly into nanoparticles. The amino acid composition of these peptides is derived from the characteristic sequences of MnSOD and MnCAT, including residues responsible for coordinating with Mn ions in the active center of the natural enzyme (H26, H74, H163, D159 in MnSOD, E36, E70, E155, H73, H188 in MnCAT), specific conserved amino acid sequences in MnSOD (K64, G68, Q141, D142; KFEDGEHY--EHAWY, involved in the formation of the α-helical structure of SOD) and MnCAT (HXXE is contained in the α-helix of CAT), and amino acids that have been reported to inhibit the activity of MnSOD (Y34, Q143, W161, E162) or MnCAT (Y42, F2) after mutation. The hydrophobic residues in the peptides simulate the hydrophobic environment of the active sites of natural MnSOD and MnCAT enzymes, promote self-assembly into nanoparticles through hydrophobic interactions, and then synthesize a series of self-assembled Mn-based peptide nanozymes with SOD-CAT cascade activity through the above method.

[0035] The optimal active sequence was screened by comparing their catalytic activities, and the results showed that after the synthesis of the nanozyme, the peptide sequence containing 32.5% α-helical amino acids showed higher catalytic activity (e.g. Figure 1 After screening, the optimal sequence PepNzyme based on the polypeptide sequence L-3 was obtained. SOD-CAT , which has the strongest SOD and CAT catalytic activity ( Figure 1 ). Self-assembled PepNzyme SOD-CATThe catalytic activity of the SOD-like enzyme reached 10,000 U / mg, and the catalytic activity of the CAT-like enzyme reached 70,000 U / mg, which are 7 times and 23 times that of the natural SOD enzyme (Sigma, S8160, 1500 U / mg) and CAT enzyme (Sigma, C9322, 3000 U / mg), respectively. Transmission electron microscopy (TEM) analysis showed that PepNzyme SOD-CAT The nanoparticles have an irregular spherical morphology, are uniformly distributed, and have a diameter of approximately 52.2 nm ( Figure 2 ). Circular dichroism (CD) spectroscopy showed that the secondary structure of the nanoparticles was composed of two structures with opposite polarities: α-helix and β-fold ( Figure 3 ), its structure remains unchanged before and after atomization ( Figure 3 ). These findings suggest that PepNzyme SOD-CAT A tertiary structure of the α / β (mixed fragments of α helix and β sheet) type is formed.

[0036] X-ray diffraction (XRD) analysis showed that PepNzyme SOD-CAT It has an amorphous structure, which may be due to the complex interaction between manganese ions, porphyrin rings and amino acids ( Figure 4 ). UV-visible spectroscopy (UV-vis) showed that Mn appeared after the synthesis of nanozymes. 3+ The characteristic peak of Figure 5 The presence of a host-guest interaction in the peptide nanozyme system was confirmed by the gradual appearance of a 430-440 nm peak within 0.5 to 5 hours, confirming that Mn 3+ - Formation of porphyrin-amino acid ligands ( Figure 5 ). The high-resolution Mn 2p spectrum shows two main peaks at 641.98 eV and 653.5 eV, corresponding to the Mn 2p3 / 2 and Mn 2p1 / 2 orbitals, respectively ( Figure 6 ).

[0037] Raman spectroscopy further confirmed the PepNzyme SOD-CAT Mn-N and Mn-O coordination bonds are formed in the reaction, which is consistent with the coordination bonds found in natural SOD and CAT enzymes. These Mn-O coordination bonds include COO-Mn and Mn-O-Mn ( Figure 8 ). At 636 cm -1 and 1244 cm -1 The asymmetric Mn-O-Mn stretching vibration peak was identified at . The Mn-N coordination bond originates from the nitrogen atom on the porphyrin ring and the C=N bond in histidine ( Fig. 9). Two peaks were observed in the NMR spectrum, located at 2.71 and 2.87 ppm, accounting for 85.65% of the signal, indicating that a single adjacent histidine H atom participated in the Mn coordination. In addition, a peak located at 7.88 ppm, accounting for 14.35%, was attributed to the 2β-H on the porphyrin ring. Therefore, the molar ratio of the porphyrin ring and the histidine ring involved in the Mn-N coordination was determined to be 1:6 ( Figure 7-Figure 9 ).exist 13 In the C NMR spectrum, proton signals corresponding to RCOO-Mn were observed at 36.51 and 31.40 ppm, and a peak was observed at 163.48 ppm, indicating the formation of Mn-O( Fig. 9 ). These findings confirmed that the porphyrin ring and the histidine pyrazole ring formed a complex with Mn during the synthesis of PepNzyme. The resulting Mn(3+ / 2+)-porphyrin-amino acid complex successfully mimicked the activity of MnSOD (Mn-N, Mn-O, Mn 3+ ) and MnCAT (Mn-O-Mn, COO-Mn, containing Mn 2 + and Mn 3+ The coordination structure in the binuclear manganese complex provides a solid reference for structural simulation ( Figure 4-Figure 9 ).

[0038] Example 2 Verification of cascade enzyme activity at the cellular level of self-assembling polypeptide nanozymes method: hACE2-293T cells were pretreated with 2 mM superoxide dismutase (SOD) inhibitor diethyl dithiocarbonate (DDC) or 500 μM catalase (CAT) inhibitor 3-amino-1,2,4-triazole (3AT) and then incubated with PepNzyme SOD -CAT Incubate for 4 hours. Cells were incubated with CCK-8 for 20 minutes to determine cell viability. 2 O 2 To enhance the comprehensive removal ability, 300 μM HO was used to 2 O 2 Cells were pretreated for 4 hours and then treated with different concentrations of PepNzyme SOD-CAT Incubate with cells for 2 hours and detect cell viability using CCK-8 kit to characterize PepNzyme SOD-CAT protective effect.

[0039] hACE2-293T and A549 cells were cultured. ROS were generated by adding glutamate and glycine (G, concentration of 40 / 8 μM). SOD-CATAfter incubation with cells, the fluorescent probe 20,70-dichlorofluorescein diacetate (DCFH-DA) was added, and the fluorescence intensity was analyzed by flow cytometry.

[0040] result When cells were treated with the superoxide dismutase (SOD) inhibitor diethyl dithiocarbonate (DDC) or the catalase (CAT) inhibitor 3-amino-1,2,4-triazole (3AT), a large amount of superoxide anions or H 2 O 2 , and reduced cell viability ( Fig.10 In addition, the self-assembled PepNzyme SOD-CAT Both non-targeted PepNzyme and 2 O 2 Toxicity to cells, while targeting PepNzyme SOD-CAT Better recovery effect ( Fig.10 As a positive control, H 2 O 2 After 4 h of incubation, ROS production was induced and cell viability was reduced. We then used 2',7'-dichlorodihydroxyfluorescein diacetate (DCFH-DA) to study the PepNzyme SOD-CAT Effects of non-targeted PepNzyme on intracellular ROS levels. The toxic effects caused by glutamate / glycine (G) led to a significant increase in intracellular ROS levels, while after the addition of PepNzyme SOD-CAT After that, the intracellular ROS level returned to normal ( Fig.10 d), which indicates that PepNzyme SOD-CAT It can effectively protect cells from ROS damage through the cascade of SOD and CAT enzyme catalytic activities.

[0041] Example 3 Verification of cascade targeting ability of self-assembling polypeptide nanozymes method: 1. Detection of the binding between the targeting peptide of self-assembling peptide nanozyme and cell surface receptors Fluorescein-labeled PepNzymes were incubated with hACE2-293T, a human embryonic kidney cell line expressing high levels of hACE2, at 4°C for 20 minutes. The cell nucleus and cell membrane were stained with Hoechst and Dil, and the distribution of FITC fluorescence on the cell membrane was detected using a laser confocal microscope (CLSM, Leica, Germany) to characterize the PepNzyme SOD-CAT targeted binding ability.

[0042] 2. Cellular uptake and intracellular distribution of self-assembling peptide nanozymes Fluorescein-labeled PepNzymes were co-cultured with hACE2-293T cells at 37°C for 6 hours. SOD-CAT Co-localization of PepNzyme with lysosomes or mitochondria at different time points was verified by laser confocal microscopy SOD-CAT Distribution in cells. Cells were fixed and images were acquired under a transmission electron microscope.

[0043] 3. In vivo targeting and distribution detection of self-assembling peptide nanozymes A pulmonary fibrosis model was constructed using transgenic mice with high expression of the human ACE2 gene to detect the targeting and distribution of self-assembling peptide nanozymes in vivo. The main organs were removed 4 or 24 hours after aerosol administration of Cy5.5-labeled self-assembling peptide nanozymes, and the fluorescence distribution intensity and tissue enrichment in lung lesions were detected using a bioluminescence imaging system. The Mn content in each organ was detected by ICP-MS 1, 7, and 30 days after administration. The targeting of self-assembling peptide nanozymes was characterized at the tissue level by immunofluorescence staining. Lung tissue samples were removed 4 hours after administration, fixed with paraformaldehyde, and 25 μm frozen sections were obtained. Laser confocal immunofluorescence imaging was used to detect the co-localization of Cy5.5-labeled self-assembling peptide nanozymes and ACE2 in lung tissue.

[0044] 4. Detection of tissue apoptosis level A pulmonary fibrosis model was constructed using transgenic mice with high expression of the human ACE2 gene, and lung tissue was extracted after aerosol administration of self-assembled peptide nanozymes. The TUNEL cell apoptosis detection kit was used to determine the apoptosis of lung tissue cells.

[0045] result PepNzyme was verified by confocal laser scanning microscopy (CLSM). SOD-CAT The results showed that after incubation at 4°C, the targeted self-assembled PepNzyme SOD-CAT and targeted PepNPs were able to bind to hACE2-293T cells that highly expressed the receptor ( Figure 11-Figure 12 ).

[0046] like Fig.13 As shown, FITC-labeled PepNzyme SOD-CAT The colocalization trend in lysosomes decreased over time, indicating that these peptide nanozymes can gradually escape from lysosomes. In contrast, most of the fluorescence of FITC coincided with mitochondria, and the colocalization coefficient increased at 6 h with an R value of 0.845, which was almost 40.24 times that of lysosomes (0.021). SOD-CATThe polypeptide chains are positively charged, but the lysosomes are not destroyed ( Fig.14 ). Therefore, it is speculated that the lysosomal escape of the peptide is achieved by fusion with the lysosomal membrane. SOD-CAT The polypeptide chain contains positively charged amino acids such as R, K, W, S, and E (Glu) that easily form amphipathic α-helical structures, as well as acid-sensitive amino acids such as W and E. SOD-CAT The acid-sensitive amino acids of β-lactamase mediate changes in its secondary structure in the acidic environment of lysosomes, thereby promoting its membrane fusion and lysosomal escape.

[0047] The designed polypeptide chain contains an aromatic-cationic motif, which can be considered as an amino acid sequence that targets mitochondria and helps to transport PepNzyme SOD-CAT In addition, transmission electron microscopy (TEM) confirmed that PepNzyme SOD -CAT After treatment, the number of mitochondria increased ( Fig.14 ). It restored the activity state and number of mitochondria to the same level as the normal cell control group. Therefore, it is speculated that by using ACE2-targeting peptide-mediated endocytosis, pH-sensitive secondary structure conversion strategy to achieve intracellular lysosomal escape and subsequent accumulation in mitochondria, PepNzyme SOD-CAT It can restore the vitality of cells and thus resist the outbreak of ROS.

[0048] To evaluate its feasibility of inhalation and ensure that it reaches the lungs accurately, we prepared Cy5.5-labeled PepNzyme SOD-CAT and non-targeted controls. SOD-CAT PepNzymes and non-targeted controls were dispersed and administered to a pulmonary fibrosis (PF) mouse model by inhalation. To verify whether these PepNzymes could be delivered to hACE2-overexpressing lung tissue, we performed immunofluorescence staining of alveolar tissue sections by CLSM. The results showed that PepNzymes were delivered to hACE2-overexpressing lung tissue by inhalation. SOD-CAT After delivery to the bleomycin-induced PF mouse model, the fluorescence intensity was the highest ( Fig.15 ). These results confirm that PepNzyme SOD-CAT Able to specifically target pathological lung tissue.

[0049] To evaluate the biodistribution of different formulations, the mean fluorescence intensity of major organs (such as lungs) collected from bleomycin-treated mice was measured by in vitro fluorescence imaging at 4 or 24 hours after administration of PepNzymes. Fig.17 As shown, PepNzyme SOD-CATThe fluorescence signal of both the targeted and non-targeted controls decreased in the lungs, indicating that the nanoparticles crossed the lung barrier. SOD-CAT It mainly accumulated in the bronchi 4 hours after administration and began to spread to the parenchymal tissue at 24 hours. However, a significant decrease in the signal of the non-targeted group was observed in the bronchi, suggesting that the non-targeted group was degraded and / or cleared more quickly in the lungs. Quantitative analysis of the whole lung also showed that PepNzyme SOD-CAT At each time point, a higher signal ( Fig.16 ). PepNzyme SOD-CAT The selectivity helps to accurately deliver the drug to the lungs.

[0050] In addition, inhaled administration allows for faster and more efficient accumulation of PepNzymes in the lungs than intravenous (iv) administration. Figure 16-Figure 17 We observed that PepNzyme SOD-CAT Distribution to other major organs including heart, liver, spleen, and kidneys. Inhaled administration accumulates less in other organs but more in the lungs than iv injection ( Fig.17 However, iv-administered PepNzyme was detected in the liver and kidneys. SOD-CAT Moreover, since nanoparticles can be absorbed by the mononuclear phagocytic system and accumulate in the liver, those that accumulate in the kidneys may be associated with renal excretion. SOD-CAT Mice with PD-L1 expression showed stronger fluorescence in lung tissue and weaker fluorescence in other major non-lung organs ( Fig.17 ). These data suggest that after inhalation administration, PepNzyme SOD-CAT The accumulation is greater than after intravenous injection, and the targeting peptide helps to direct PepNzyme SOD-CAT Targets specifically to the lungs, thereby reducing PepNzyme SOD-CAT Accumulation in other major organs. ICP-MS analysis ( Fig.18 ) showed that after 30 days of inhalation or injection, PepNzyme in mice SOD-CAT This can be confirmed by analyzing the Mn content in various tissues.

[0051] In addition, according to the TUNEL staining results of lung tissue sections, PepNzyme SOD-CAT The number of apoptotic lung cells in mice with pulmonary fibrosis treated with Fig.19 ).

[0052] Example 4 Evaluation of the therapeutic effect of pulmonary fibrosis method: Transgenic mice with high expression of human ACE2 gene were injected with 5 mg / kg bleomycin through the trachea to establish PF model. The mice were randomly divided into 5 groups (PepNzyme SOD-CAT group, non-targeted group, pirfenidone group, model group and normal group, 5 mice in each group). SOD-CAT The pirfenidone group and the non-targeted control group were administered by aerosol, and the pirfenidone treatment group was administered orally. The therapeutic effects were evaluated on the 5th and 30th days, respectively. Lung tissues were collected to observe the appearance, color, texture, edema, and necrosis range of inflammatory indicators. The mice were euthanized by overdose of pentobarbital, and lung tissues, blood, and bronchoalveolar lavage fluid (BALF) were collected. The permeability of lung tissue was analyzed by Evans blue staining; the anti-inflammatory effect was evaluated by detecting the level of C-reactive protein (CRP); lungs were harvested on the 30th day, and lung tissue sections were stained with HE, Sirius red, and Masson to observe collagen fiber deposition; the anti-fibrotic effect was evaluated by immunohistochemical detection of collagen I (COL1A1) and α-smooth muscle actin (α-SMA) in lung tissue. After the mice were killed, fresh lung tissues on both sides were collected and weighed (wet weight). Then, the lung tissues were placed in an oven at 60°C for 48 hours and weighed (dry weight). The wet / dry weight ratio of lung tissue was calculated by weighing before and after.

[0053] result PepNzyme SOD-CAT The PF mouse model was given 0.2 mg / kg by nebulization on days 0, 2, and 4, respectively, and the non-targeted control group was given Pirfenidone by oral administration (150 mg / kg per day for 21 consecutive days) ( Fig. 20 ). Observe the appearance of the lungs. Normal lungs are uniform in color, round and smooth. On the 5th day after the model was established, the lungs were uneven in color, with obvious edema and swelling due to congestion, edema and necrosis caused by inflammation. PepNzyme SOD-CAT It is able to effectively clear inflammation, and the appearance of the lungs returns to normal after 5 days. After 30 days, the lungs have uneven color, uneven texture, or nodular masses due to fibrosis and necrosis. PepNzyme SOD-CAT It was able to effectively reverse the fibrosis process, with the lung appearance returning to normal after 5 days and no PF symptoms appearing after 30 days ( Fig.21 ). Oral administration of high doses of pirfenidone (150 mg / kg per day for 21 days) partially alleviated the pathological changes associated with PF. However, this effect was still inferior to that of PepNzyme SOD-CAT Effect of inhalation (0.2 mg / kg per day for 2 days). Bronchoalveolar lavage fluid (BALF) samples were collected from mice 5 or 30 days after inhalation treatment to detect inflammatory responses. SOD-CATIn the treatment group, the CRP level in BALF decreased significantly, but did not increase after 30 days ( Fig. 22 ).

[0054] To evaluate the pathological changes, we performed hematoxylin-eosin (H&E), Masson and Sirius red staining, and applied the lung injury scoring system. H&E staining showed that the mice in the model group had severe lung lesions on day 5, accompanied by obvious inflammatory infiltration and multiple dense nodules, and thickening of the alveolar wall ( Fig.23 ). In contrast, PepNzyme SOD-CAT The alveolar structure of the treatment group was clearly visible, and the lung inflammation score was significantly reduced ( Fig.24 The results of H&E staining on day 30 showed that PepNzyme SOD-CAT Treatment strongly attenuated bleomycin-induced fibrosis and preserved alveolar epithelial architecture ( Fig.23 , Fig.25 , Fig.26 In addition, Masson staining, Ashcroft score analysis, and Picrosirius red staining showed that extensive fibrosis occurred in the lung tissues of the non-targeted control group, pirfenidone-treated group, and model mice ( Figure 25-28 However, inhaled PepNzyme SOD-CAT After inhalation of PepNzyme, collagen deposition and parenchymal destruction were significantly reduced. Immunofluorescence staining images showed that bleomycin induced the expression of COL1A1 and α-SMA in the lung parenchyma, while inhaled PepNzyme SOD-CAT After that, its expression was significantly reduced ( Fig.29 ). To study PepNzyme SOD-CAT The effectiveness of improving lung injury and vascular leakage was assessed by Evans blue dye to assess pulmonary vascular permeability ( Fig.30 ). After bleomycin injection, extravasation of Evans blue dye outside the pulmonary vasculature was significantly increased. Compared with non-targeted control treatments that did not show good efficacy, PepNzyme SOD-CAT The results showed that PepNzyme SOD-CAT Significantly reduced lung wet / dry weight ratio ( Fig.31 ).

[0055] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0056] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0057] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A self-assembling polypeptide nanozyme, characterized in that: include: Peptide scaffolds with targeting and nanozyme affinity peptide sequences, including: The main body, whose amino acids have characteristic sequences derived from natural enzymes, is composed of amino acids coordinating the active center of natural enzymes and amino acids participating in the formation of α-helical structures in the conserved sequences of natural enzymes. Contains a functional motif of hydrophobic peptides, which self-assemble into peptide nanoparticles using the hydrophobic interactions of amino acids; A nanozyme, coordinated and bound to the central coordinating amino acid; The targeting peptide is located at the N-terminus of the polypeptide sequence and is displayed on the outer surface of the self-assembled polypeptide nanoparticles.

2. The self-assembling polypeptide nanozyme according to claim 1, characterized in that The main motif includes 5-50 amino acids and is an amino acid sequence that can form a tertiary structure.

3. The self-assembling polypeptide nanozyme according to claim 2, characterized in that The amino acid sequence of the motif of the subject is QDWEH, QDEWEH, KFHGGGDKNHQDHHDKWEH, LKHLDGLRLHGKDNQDHHEK, KFEDGEHYHRHQDSWEH, KFEDGEAYHRHQDSWEH, KFEDGEHYARHQDSWEH, KFEDGEHYHRAQDSWEH, KFEDGEHYHRHQDSWEA, KFEDGEHYHRHQASWEH, KFEDGEHAHRHQDSWEH, KFEDGEHYHRHADSWEH, KFEDGEHYHRHQDSAEH or KAEDGEHYHRHQDSWEH.

4. The self-assembling polypeptide nanozyme according to claim 2, characterized in that The main motif comprises 5-17 amino acids.

5. The self-assembling polypeptide nanozyme according to claim 1, characterized in that The hydrophobic peptide-containing functional motif comprises 5-50 amino acids.

6. The self-assembling polypeptide nanozyme according to claim 5, characterized in that The hydrophobic amino acids containing the functional motif of the hydrophobic peptide are one or any combination of A, F, I, L, M, P, V, W, and Y, or a hydrophobic polypeptide with biological function.

7. The self-assembling polypeptide nanozyme according to claim 6, characterized in that: The functional motif containing the hydrophobic peptide is a combination sequence of AYYLLY, WLFLL, AYYLQYAYYL, PALWLFLVLL or AWYFWAFLWL.

8. The self-assembling polypeptide nanozyme according to claim 1, characterized in that: The nanozyme is a complex formed by metal ions and amino acids and / or cofactors, metals, or metal oxides.

9. The self-assembling polypeptide nanozyme according to claim 8, characterized in that The cofactor is porphyrin, macrocyclic polyamine or ammonia.

10. Use of the self-assembling polypeptide nanozyme according to any one of claims 1 to 9 in the preparation of a nebulized inhalation therapeutic drug for lung diseases.

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