Biosafe and degradable cerium-based tannic acid nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a biologically safe and degradable cerium-based tannic acid nano-enzyme as well as a preparation method and application thereof. The nano-enzyme can be administrated through a non-invasive nasal cavity. In an area with excessive ROS, thioketone on CeTA-k1tkP is destroyed, PEG is removed, hydrogen bonds of an amino acid sequence are allowed to form beta-sheet, the beta-sheet is accumulated at an inflammation part, and a fiber or network structure is formed. Meanwhile, CeTA-k1tkP shows enzyme-like capacity, ROS is effectively eliminated, inflammation is effectively relieved, and systemic immunosuppression is avoided at the same time. In an influenza A (Flu A) virus or Sendai virus (SeV) mediated viral pneumonia model, CeTA-k1tkP can generate responsive deformation in inflammatory lung tissues, so that the nano-enzyme effectively decomposes ROS (reactive oxygen species). In addition, the CeTA-k1tkP can relieve bacterial inflammation and reduce tissue damage in a mouse viral pneumonia model with secondary bacterial infection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a biosafe and degradable cerium-based tannic acid nanozyme and a preparation method and application thereof. Background Art

[0002] Pneumonia is a widespread disease caused by viral and bacterial infections. Respiratory viruses can be transmitted through aerosols and droplets, and can easily cause clustered infections. Respiratory viruses enter cells through endocytosis, accumulate in endosomes, and trigger Nox2 oxidase activity, resulting in the production of a large amount of reactive oxygen species (ROS). In order to inhibit viral replication, clinical treatment usually uses antiviral drugs, including small molecule compounds, peptides, and antibodies. At present, only nucleoside analogs targeting viral DNA / RNA polymerase and compounds targeting host proteins can be used as broad-spectrum antiviral drugs to treat a wide range of viruses. However, for emerging viruses, their sequences may not be determined in a short period of time, which makes it more difficult to quickly develop specific drugs and protease inhibitors. Therefore, it is imperative to establish a therapeutic strategy for broad-spectrum viral pneumonia.

[0003] Downregulation of ROS levels in inflammatory areas is essential for alleviating inflammation, protecting tissues from oxidative damage, and inhibiting viral replication. Redox balance in normal cells is maintained by a series of enzymatic antioxidants, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione peroxidase (GSH-Px). However, the application of natural enzymes in diseases is full of challenges and limitations. Natural enzymes are usually composed of protein and RNA molecules, which are easily affected by environmental variables and easily lose their activity under pathological conditions. In order to address the many shortcomings of natural enzyme antioxidants, people have designed and constructed "nanozymes" that can replace the antioxidant function of natural enzymes. It has been reported that nanozymes have been used to treat many inflammation-related diseases, including neuroinflammation, infectious inflammation, and osteoarthritis. However, due to the complexity of internal inflammation, precise delivery and accumulation at the inflammatory site for the treatment of viral pneumonia is challenging. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a biosafe and degradable cerium-based tannic acid nanozyme and its preparation method and application, which has the ability to respond to the aggregation of pathological areas where ROS are produced in large quantities, thereby enhancing the catalytic activity and the ability to scavenge electron free radicals. It has a broad spectrum of effects on inflammatory diseases and can be administered in a variety of ways. It is expected to play a therapeutic role in COVID-19, sepsis, enteritis and arthritis.

[0005] The present invention is achieved by a method for preparing a biosafe and degradable cerium-based tannic acid nanozyme, comprising the following steps: 1) Preparation of CeTA nanozyme: CeTA nanozyme was prepared by hydrothermal method using tannic acid and cerium nitrate hexahydrate as raw materials, wherein the mass ratio of tannic acid to cerium nitrate hexahydrate was 1:32; 2) preparing alkyl-modified CeTA nanozyme: using the CeTA nanozyme prepared in step 1), methacrylic acid (MAA), propyl methacrylate (PMA), divinylbenzene (DVB) and isobutyronitrile as raw materials, preparing alkyl-modified CeTA nanozyme by distillation precipitation method; 3) Preparation of ROS-responsive CeTA-k1tkp nanozyme: CeTA-k1tkp nanozyme was prepared using the alkyl-modified CeTA nanozyme prepared in step 2) and N3-KGGGKLVFF-tk-mPEG (SEQ ID NO.1) polypeptide.

[0006] The diameter of the CeTA nanozyme is 30 to 40 nanometers; the diameter of the CeTA-k1tkp nanozyme is 100 to 300 nanometers.

[0007] Step 1) Preparation method of CeTA nanozyme: According to the mass ratio of tannic acid to cerium nitrate hexahydrate of 1:32, tannic acid and cerium nitrate hexahydrate solid powders are weighed and dissolved in deionized water, reacted at a temperature of 30-100°C for 1-12 hours, and dried in an oven at 30-60°C overnight to obtain CeTA nanozyme.

[0008] Step 2) Preparation method of alkyl-modified CeTA nanozyme: (1) Weigh the CeTA nanozyme, methacrylic acid, propyl methacrylate and isobutyronitrile prepared in step 1), dissolve them in acetonitrile and stir them evenly. The mass ratio of CeTA nanozyme, methacrylic acid, propyl methacrylate and isobutyronitrile is 1:2:0.5:0.35:0.025; (2) After the solution is mixed evenly, it is added into a reactor equipped with a condensation reflux device, and the reaction is carried out at a reflux temperature of 60 to 100°C for 1 to 12 hours. After the reaction is completed, the nanoparticles are collected by centrifugation and dried in an oven at 30 to 60°C overnight to obtain alkyl-modified CeTA nanozyme.

[0009] Step 3) Preparation method of ROS-responsive CeTA-k1tkp nanozyme: Weigh the alkyl-modified CeTA nanozyme and N3-KGGGKLVFF-tk-mPEG prepared in step 2) and dissolve them in a NaHCO3 solution with a pH of 8.0, and stir at room temperature for 2 to 12 hours to obtain CeTA-k1tkp nanozyme. The mass ratio of alkyl-modified CeTA nanozyme: N3-KGGGKLVFF-tk-mPEG: NaHCO3 is 1:1:1, and the total concentration of NaHCO3 in the mixed solution is 14.28 mmol / mL.

[0010] The biosafe and degradable cerium-based tannic acid nanozyme prepared by the above-mentioned preparation method.

[0011] The application of the above-mentioned biosafe and degradable cerium-based tannic acid nanozyme in the preparation of drugs that promote the fusion of viral pneumonia.

[0012] The advantages and technical effects of the present invention are as follows: 1) Based on peptide-based biological self-assembly, an inflammatory response nanozyme (CeTA-K1tkP) is invented with a preparation size of 100-300 nanometers. 2) The CeTA-K1tkP nanozyme can be administered through non-invasive nasal administration. In areas where ROS are excessive, the thione on CeTA-K1tkP is destroyed and PEG is removed, eliminating 80% of the hydrophilic "barrier", allowing the hydrogen bonds of the amino acid sequence to form β-sheets, accumulate at the site of inflammation, and form a fiber or network structure. 3) CeTA-K1tkP exhibits enzyme-like ability, effectively eliminates ROS and relieves inflammation while avoiding systemic immunosuppression. 4) In the viral pneumonia model mediated by influenza A (Flu A) virus or Sendai virus (SeV), CeTA-K1tkP can undergo responsive deformation in inflamed lung tissue, allowing the nanozyme to effectively decompose ROS. 5) CeTA-K1tkP can alleviate bacterial inflammation and reduce tissue damage in a mouse viral pneumonia model with secondary bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 TEM images of CeTA nanozymes after 28 days in solutions of pH = 7.0 or pH = 4.5.

[0014] Figure 2 is the high-resolution XPS image of Ce1s and Ce 3d peaks of CeTA nanozyme.

[0015] Figure 3 is the EDS analysis result of the SEM image after self-assembly of CeTA-K1tkP.

[0016] Figure 4 is a representative TEM image of CeTA-K1tkP after adding 80 μM H2O2.

[0017] Figure 5 This is an image of CeTA nanozyme removing •OH.

[0018] Figure 6 This is an image of CeTA-K1tkP nanozyme clearing •OH.

[0019] Figure 7 CeTA nanozymes remove O2 - image.

[0020] Figure 8 CeTA-K1tkP nanozyme removes O2 - image.

[0021] Fig. 9 This is a confocal laser scanning microscopy image using a fluorescent probe (DCFH-DA) to detect cellular ROS.

[0022] Fig.10 Representative confocal laser scanning microscopy images of ROS in CeTA-K1tkP nanozymes A549 cells and BEAS-2B cells.

[0023] Fig.11 Representative H&E staining images of mouse lung sections after intranasal instillation of nanozymes in the mouse pneumonia model mediated by influenza A virus (H1N1).

[0024] Fig.12 Representative H&E staining images of mouse lung sections after intranasal instillation of nanozymes in SeV-induced acute pneumonia model mice.

[0025] Fig.13 Representative H&E staining images of mouse lung sections after modeling viral pneumonia combined with bacterial infection using SeV and Staphylococcus aureus and intranasal instillation of nanozymes in mice. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] The method for preparing the biosafe and degradable cerium-based tannic acid nanozyme of the present invention comprises the following steps: 1) Preparation of CeTA nanozyme: CeTA nanozyme was prepared by hydrothermal method using tannic acid and cerium nitrate hexahydrate as raw materials, wherein the mass ratio of tannic acid to cerium nitrate hexahydrate was 1:32; 2) preparing alkyl-modified CeTA nanozyme: using the CeTA nanozyme prepared in step 1), methacrylic acid (MAA), propyl methacrylate (PMA), divinylbenzene (DVB) and isobutyronitrile as raw materials, preparing alkyl-modified CeTA nanozyme by distillation precipitation method; 3) Preparation of ROS-responsive CeTA-k1tkp nanozyme: CeTA-k1tkp nanozyme was prepared using the alkyl-modified CeTA nanozyme prepared in step 2) and N3-KGGGKLVFF-tk-mPEG (SEQ ID NO.1) polypeptide.

[0028] The diameter of the CeTA nanozyme is 30 to 40 nanometers; the diameter of the CeTA-k1tkp nanozyme is 100 to 300 nanometers.

[0029] Step 1) Preparation method of CeTA nanozyme: According to the mass ratio of tannic acid to cerium nitrate hexahydrate of 1:32, tannic acid and cerium nitrate hexahydrate solid powders are weighed and dissolved in deionized water, reacted at a temperature of 30-100°C for 1-12 hours, and dried in an oven at 30-60°C overnight to obtain CeTA nanozyme.

[0030] Step 2) Preparation method of alkyl-modified CeTA nanozyme: (1) Weigh the CeTA nanozyme, methacrylic acid, propyl methacrylate and isobutyronitrile prepared in step 1), dissolve them in acetonitrile and stir them evenly. The mass ratio of CeTA nanozyme, methacrylic acid, propyl methacrylate and isobutyronitrile is 1:2:0.5:0.35:0.025; (2) After the solution is mixed evenly, it is added into a reactor equipped with a condensation reflux device, and the reaction is carried out at a reflux temperature of 60 to 100°C for 1 to 12 hours. After the reaction is completed, the nanoparticles are collected by centrifugation and dried in an oven at 30 to 60°C overnight to obtain alkyl-modified CeTA nanozyme.

[0031] Step 3) Preparation method of ROS-responsive CeTA-k1tkp nanozyme: Weigh the alkyl-modified CeTA nanozyme and N3-KGGGKLVFF-tk-mPEG prepared in step 2) and dissolve them in a NaHCO3 solution with a pH of 8.0, and stir at room temperature for 2 to 12 hours to obtain CeTA-k1tkp nanozyme. The mass ratio of alkyl-modified CeTA nanozyme: N3-KGGGKLVFF-tk-mPEG: NaHCO3 is 1:1:1, and the total concentration of NaHCO3 in the mixed solution is 14.28 mmol / mL.

[0032] The biosafe and degradable cerium-based tannic acid nanozyme prepared by the above-mentioned preparation method.

[0033] The application of the above-mentioned biosafe and degradable cerium-based tannic acid nanozyme in the preparation of drugs that promote the fusion of viral pneumonia.

[0034] The N3-KGGGKLVFF-tk-mPEG polypeptide of the present invention was purchased from Nanjing Peptide Industry Biotechnology Co., Ltd. The peptides containing Lys (N3) at the C-terminus were prepared by a standard Fmoc solid-phase peptide synthesis method and purified by reverse-phase high-performance liquid chromatography.

[0035] The present invention will be further described in the following examples, but the present invention is not limited thereto.

[0036] Example 1 The preparation steps of CeTA nanozyme are as follows: 1) CeTA nanozymes with a diameter of 30 to 40 nanometers were synthesized by hydrothermal reaction. During the synthesis process, 27 mg of tannic acid and 875 mg of cerium nitrate hexahydrate were weighed and dissolved in 50 mL of deionized water, and reacted at 50 °C for 3 hours.

[0037] 2) The CeTA nanozyme was then dried in an oven at 50 °C overnight. The CeTA nanozyme was characterized by TEM under acidic conditions. Figure 1 As shown. By X-ray photoelectron spectroscopy (XPS), trivalent cerium and tetravalent cerium (Ce 3+ / Ce 4+ ) is 1:2, indicating that the main antioxidant tetravalent cerium such as Figure 2 shown.

[0038] Example 2 The preparation method of alkyl-modified CeTA nanozyme is as follows: 1) Using methacrylic acid, propyl methacrylate and divinylbenzene as raw materials, alkyl-modified CeTA nanozyme was prepared by distillation precipitation copolymerization in acetonitrile. 200 mg of CeTA nanozyme prepared in Example 1 was weighed and dispersed in 40 ml of acetonitrile, and ultrasonicated for 0.5 h in a 100 mL double-necked round-bottom flask equipped with a reflux condenser. Then, methacrylic acid (0.20 mL, 430.45 mg), propyl methacrylate (0.2 mL, 115.353 mg), divinylbenzene (0.3 mL, 71.6045 mg) and isobutyronitrile (0.2 mL, 5 mg) were weighed and dissolved in acetonitrile, mixed and placed in a flask.

[0039] 2) A two-necked round-bottom flask equipped with a condensing reflux device. The polymerization temperature was increased from 25°C to the reflux temperature of the reaction mixture at 83°C within 25 minutes. Then, the copolymerization reaction was carried out under reflux conditions for 4 hours. The synthesized nanospheres were collected by centrifugation and dried in a vacuum oven at 50°C overnight to obtain alkyl-modified CeTA nanozymes.

[0040] Example 3 The preparation method of the ROS-responsive CeTA-k1tkp nanozyme is as follows: 1) 6 mg of N3-KGGGKLVFF-tk-mPEG was dissolved in 5 mL of pH 8.0 NaHCO3 solution, and then the alkyl-modified CeTA nanozyme prepared in Example 2 was added and stirred at room temperature for 2 hours to obtain CeTA-k1tkp nanozyme.

[0041] 2) To detect the self-assembly of CeTA-k1TKP in vitro, H2O2 was used to simulate an excessive ROS environment. Scanning electron microscopy (SEM) images of CeTA-k1TKP in a ROS environment were taken as follows: Figure 3 As shown and transmission electron microscopy Figure 4 The CeTA-k1tkp nanoplatform shown here twists and aggregates in an in vitro simulated inflammatory environment (80 μM H2O2). This proves that the KLVFF peptide is triggered to form a β-sheet under the ROS environment and the nanozyme begins to self-assemble.

[0042] Example 4 The steps of the active oxygen scavenging ability experiment are as follows: 1) Using DMPO as a scavenger, the ESR spectroscopy was used to study the effects of nanozymes on •OH and O2 - DMPO (0.6 mg) was dissolved in 1.3 mM H2O2 in PBS buffer. CeTA and CeTA-k1tkp were added to the solution and mixed for 5 min. The ESR amplitude intensity was used to quantify the amount of •OH. Figure 5 and Figure 6 shown.

[0043] 2) The removal of O2 by CeTA and CeTA-k1tkp nanozymes in methanol solution according to the above method - Ability to test Figure 7 and Figure 8 shown.

[0044] Example 5 The experimental steps for cell ROS detection are as follows: 1) RAW 264.7 cells were cultured in 6-well confocal plates (2 × 10 cells per well). 5 The cells were pretreated with LPS (100 ng / mL) for 4 h and then incubated with gradient concentrations of CeTA and CeTA-k1tkp for 24 h.

[0045] 2) Detect cellular ROS using a fluorescent probe (DCFH-DA) under confocal laser scanning microscopy (CLSM), such as Fig. 9 and Fig.10 shown.

[0046] Example 6 The construction and treatment experimental steps of the pneumonia infection mouse model are as follows: 1) For the viral pneumonia mouse model, mice were lightly anesthetized with ether, and the control group was gavaged with 10 μL PBS through the nose, and the other groups were instilled with the same amount of SeV (hemagglutination efficiency 1:640) or H1N1 virus (100 TCID50) through the nose to prepare SeV or H1N1 lung infection model. 48 hours after the model was established, nanozyme (1.5 μg / g) was instilled through the nose for 2 consecutive days. Representative H&E images of mouse lungs and trachea after nasal instillation of nanozyme treatment are shown in the figure. Fig.11 As shown and Fig.12 shown.

[0047] 2) For virus-bacteria co-infection pneumonia, mice were injected intranasally with 2 10 8 CFU Staphylococcus aureus. After 24 hours of model establishment, nanozymes (1.5 μg / g) were instilled into the nasal cavity for 3 consecutive days. Representative H&E images of mouse lungs and trachea after nasal instillation of nanozymes Fig.13 All aspiration manipulations were performed slowly through a 10 μL pipette tip.

[0048] The present invention is based on peptide-based biological self-assembly, and invents an inflammatory response nanozyme (CeTA-K1tkP), which responds to the aggregation of pathological areas where ROS are produced in large quantities, thereby enhancing the catalytic activity and the ability to scavenge electron free radicals. CeTA-K1tkP nanozyme has a broad spectrum of effects on inflammatory diseases and can be administered in a variety of ways. It is expected to play a therapeutic role in COVID-19, sepsis, enteritis and arthritis. In addition, CeTA nanozymes in inhalable non-invasive nanodelivery based on inflammation-triggered peptides can also be replaced by other catalytic nanomaterials or other therapeutic drugs such as interferon, antibiotics, hormones, etc., to optimize the delivery targeting of therapeutic drugs and effectively eliminate their potential toxic side effects. Therefore, the inhalable inflammation-triggered self-assembled nanozyme delivery strategy provides a promising paradigm for the treatment of various deep inflammations, such as pneumonia.

[0049] Those skilled in the art can achieve the final preparation technology by appropriately changing the conditions and routes by referring to the contents of this article. Although the method and preparation technology of the present invention have been described by preferred embodiments, relevant technicians can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention.

Claims

1. A method for preparing a biosafe and degradable cerium-based tannic acid nanozyme, characterized in that: The following steps are involved: 1) Preparation of CeTA nanozyme: CeTA nanozyme was prepared by hydrothermal method using tannic acid and cerium nitrate hexahydrate as raw materials, wherein the mass ratio of tannic acid to cerium nitrate hexahydrate was 1:32; 2) preparing alkyl-modified CeTA nanozyme: using the CeTA nanozyme prepared in step 1), methyl methacrylate MAA, propyl methacrylate PMA, divinylbenzene DVB and isobutyronitrile as raw materials, preparing alkyl-modified CeTA nanozyme by distillation precipitation method; 3) Preparation of ROS-responsive CeTA-k1tkp nanozyme: CeTA-k1tkp nanozyme was prepared using the alkyl-modified CeTA nanozyme prepared in step 2) and N3-KGGGKLVFF-tk-mPEG (SEQ ID NO.1) polypeptide.

2. The method for preparing the biosafe and degradable cerium-based tannic acid nanozyme according to claim 1, characterized in that: The diameter of the CeTA nanozyme is 30 to 40 nanometers; the diameter of the CeTA-k1tkp nanozyme is 100 to 300 nanometers.

3. The method for preparing the biosafe and degradable cerium-based tannic acid nanozyme according to claim 1, characterized in that: Step 1) Preparation method of CeTA nanozyme: According to the mass ratio of tannic acid to cerium nitrate hexahydrate of 1:32, tannic acid and cerium nitrate hexahydrate solid powders are weighed and dissolved in deionized water, reacted at a temperature of 30-100°C for 1-12 hours, and dried in an oven at 30-60°C overnight to obtain CeTA nanozyme.

4. The method for preparing the biosafe and degradable cerium-based tannic acid nanozyme according to claim 1, characterized in that: Step 2) Preparation method of alkyl-modified CeTA nanozyme: (1) Weigh the CeTA nanozyme, methacrylic acid, propyl methacrylate and isobutyronitrile prepared in step 1), dissolve them in acetonitrile and stir them evenly. The mass ratio of CeTA nanozyme, methacrylic acid, propyl methacrylate and isobutyronitrile is 1:2:0.5:0.35:0.025; (2) After the solution is mixed evenly, it is added into a reactor equipped with a condensation reflux device, and the reaction is carried out at a reflux temperature of 60-100 °C for 1-12 hours. After the reaction is completed, the nanoparticles are collected by centrifugation and dried in an oven at 30-60 °C overnight to obtain alkyl-modified CeTA nanozyme.

5. The method for preparing the biosafe and degradable cerium-based tannic acid nanozyme according to claim 1, characterized in that: Step 3) Preparation method of ROS-responsive CeTA-k1tkp nanozyme: Weigh the alkyl-modified CeTA nanozyme and N3-KGGGKLVFF-tk-mPEG prepared in step 2) and dissolve them in a NaHCO3 solution with a pH of 8.0, and stir at room temperature for 2 to 12 hours to obtain CeTA-k1tkp nanozyme. The mass ratio of alkyl-modified CeTA nanozyme: N3-KGGGKLVFF-tk-mPEG: NaHCO3 is 1:1:1, and the total concentration of NaHCO3 in the mixed solution is 14.28 mmol / mL.

6. A biosafe and degradable cerium-based tannic acid nanozyme prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the biosafe, degradable cerium-based tannic acid nanozyme as described in claim 6 in the preparation of a drug that promotes the fusion of viral pneumonia.

Images