Preparation of cationic nano-material and application of cationic nano-material in treatment of respiratory tract inflammation

Abstract

The invention discloses preparation of a cationic nano-material and application of the cationic nano-material in treatment of respiratory tract inflammation. The preparation method of the cationic nano material comprises the following steps: S1, connecting a compound with ortho-dihydroxyl and carboxyl at the same time with a boron nanosheet with hydroxyl through a boron esterification reaction, and then modifying the boron nanosheet with a cationic polymer with amino through an amidation reaction; or modifying the cationic polymer with the ortho-dihydroxyl group on the boron nanosheet with the hydroxyl group through a boron esterification reaction; and S2, dialyzing the boron nanosheet treated in the step S1 to obtain the cationic nano material. The cationic nano material provided by the invention has good biological safety, can target cfDNA / NETs / EETs, ROS / RNS and bacterial infection at the same time, is highly enriched in inflammatory parts in vivo, remarkably reduces the generation of airway mucus, and effectively relieves airway inflammation.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomedicine, and more specifically, relates to the preparation of a type of cationic nanomaterial and its application in treating respiratory inflammation. Background Art

[0002] Allergic rhinitis, chronic sinusitis, asthma and other respiratory tract inflammations affect millions of people worldwide, causing a significant medical burden on society. Commonly used clinical drugs (such as glucocorticoids) are restricted in their use due to their limited efficacy and significant side effects. Therefore, it is of great significance to develop new treatment strategies to minimize systemic side effects while achieving ideal therapeutic effects.

[0003] Patients with severe respiratory inflammatory diseases often have extensive neutrophil and eosinophil infiltration in the airway tissues, and this inflammatory feature is closely related to the patient's glucocorticoid resistance. Granulocytes can respond to external stimuli (such as bacterial infection, cfDNA released by damaged airway epithelium, etc.) to form extracellular traps (NETs / EETs). Recombinant human DNA enzyme (DNase) has been shown to relieve airway inflammation and reduce mucus production by clearing NETs / EETs. However, due to the short effective period and significant side effects of DNase, it is difficult to be used in the clinical treatment of diseases.

[0004] Recently, the progress of nanomedicine has provided new methods for clinical diagnosis and treatment. Many studies have shown that nanomaterials can relieve inflammation by removing excess cfDNA / NETs / EETs. However, the therapeutic effect of nanomedicine in removing cfDNA is still not ideal. For example, the increase of reactive oxygen and reactive nitrogen (ROS / RNS) at the site of inflammation and bacterial infection can cause continuous damage to the airway epithelium. Therefore, the development of multifunctional nanomaterials that can simultaneously respond to multiple danger signals in pathological tissues is crucial for the effective treatment of airway inflammation. Summary of the invention

[0005] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide a method for preparing cationic nanomaterials. The cationic nanomaterials prepared by the preparation method have good biosafety, can simultaneously target cfDNA / NETs / EETs, ROS / RNS and prevent bacterial infection, are highly enriched in inflammatory sites in the body, significantly reduce airway mucus production, and effectively relieve airway inflammation.

[0006] The second object of the present invention is to provide a cationic nanomaterial prepared by the above preparation method.

[0007] The third object of the present invention is to provide the use of the above cationic nanomaterials in the preparation of drugs for treating respiratory inflammation or other inflammatory diseases.

[0008] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0009] The present invention claims a method for preparing a cationic nano material, comprising the following steps:

[0010] S1. connecting a compound having both ortho-dihydroxyl and carboxyl groups to a boron nanosheet having a hydroxyl group by boroesterification, and then modifying a cationic polymer having an amino group on the boron nanosheet by amidation; or

[0011] The cationic polymer with ortho-dihydroxyl groups was modified on the boron nanosheets with hydroxyl groups through borylation reaction;

[0012] S2. The boron nanosheets treated in step S1 are dialyzed to obtain cationic nanomaterials;

[0013] The cationic polymer with amino groups includes polyamidoamine dendrimers; and the cationic polymer with vicinal dihydroxyl groups includes hyperbranched amino polyglycerol.

[0014] The cationic nanomaterial prepared by the present invention has good biocompatibility, can absorb cfDNA, inhibit the generation of NETs / EETs, highly enrich in the inflammatory site in the body, significantly reduce the production of airway mucus, and effectively relieve airway inflammation; in addition, it also has both antibacterial and antioxidant capabilities, and has multiple inflammatory signal targeting effects. The cationic nanomaterial provided by the present invention can be used as a multifunctional nanoplatform for the treatment of respiratory inflammatory and other inflammatory diseases.

[0015] Preferably, the size of the cationic nano material is 80-700nm. Further preferably, the size of the cationic nano material is 200-300nm. Under this preferred range, the cationic nano material has a more suitable size, can be better enriched in the inflammation site, prolong the in vivo residence time of the material, and can better play an anti-inflammatory effect. Specifically, the size of the cationic nano material can be 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, etc., or the interval range formed by any of the above numerical values, such as 80-300nm, 300-700nm, 80-100nm, 200-300nm, 500-700nm, etc., the present invention is not limited to this. More specifically, the test method of the size of the cationic nano material is: dynamic light scattering (DLS). More specifically, the size of the cationic nano material refers to the diameter of the cationic nano material.

[0016] Preferably, in some embodiments, the boron nanosheets after step S1 treatment can be ultrasonically treated to obtain cationic nanomaterials of different sizes. The specific processing parameters of the ultrasonic treatment, such as power, ultrasonic treatment on / off cycle, and total ultrasonic time can be adjusted as appropriate. In some embodiments, the power of the ultrasonic treatment is 300-600W, the on / off cycle of the ultrasonic treatment is 5s / 5s, and the total ultrasonic treatment time can be set to 5-30min. More specifically, the total ultrasonic treatment time can be 30min, 15min, and 5min, respectively, to obtain cationic nanomaterials of three sizes of 80-100nm, 200-300nm, and 500-700nm, respectively.

[0017] Preferably, the Zeta potential of the cationic nanomaterial is +15mV to +35mV. Further preferably, the Zeta potential of the cationic nanomaterial is +19mV ​​to +33.2mV. Specifically, the Zeta potential of the cationic nanomaterial can be +15mV, +17mV, +19mV, +21mV, +23mV, +25mV, +27mV, +39mV, +41mV, +43mV, etc., or an interval range formed by any of the above values, such as +15mV to +30mV, +21mV to +29mV, etc., and the present invention is not limited thereto.

[0018] Preferably, in step S1, the boron nanosheets with hydroxyl groups can be obtained by purchasing commercial products or preparing the boron nanosheets with hydroxyl groups by conventional methods in the art. Preferably, in some embodiments, the boron nanosheets with hydroxyl groups are obtained by hydrolyzing metal borides. Preferably, the metal boride is selected from one or more of aluminum boride, beryllium boride, and magnesium boride. More preferably, the metal boride is selected from magnesium boride.

[0019] More specifically, in some embodiments, the preparation method of boron nanosheets with hydroxyl groups is: reacting magnesium boride in pure water, maintaining a uniform mixing state during the reaction, and after the reaction, centrifuging and dialyzing the supernatant to obtain the boron nanosheets with hydroxyl groups.

[0020] Preferably, the reaction time in pure water is 96-120 hours.

[0021] Preferably, ultrasonic treatment may be performed during the reaction to shorten the hydrolysis reaction process.

[0022] Preferably, during the reaction, the reactants can be mixed by shaking or oscillating, etc., which are conventionally used in the art to keep the reactants in a mixed state.

[0023] Preferably, the centrifugation conditions are: 1000-2000 rpm, 40-60 min. More specifically, the centrifugation conditions are: 1400-1600 rpm, 40-50 min. The centrifugation of the supernatant is performed at least once; in some embodiments, the centrifugation of the supernatant is repeated twice.

[0024] Preferably, the molecular weight of the dialyzed is 1000-10000D, and the dialysis time is 20-30h. More preferably, the molecular weight of the dialyzed is 1000-2000D.

[0025] In some embodiments, the specific operation of step S1 is: mixing boron nanosheets with hydroxyl groups and a compound with both vicinal dihydroxyl groups and carboxyl groups; then adding cationic polymers with amino groups and catalysts, and modifying the cationic polymers with amino groups on the boron nanosheets through an amidation reaction.

[0026] Preferably, the compound having both vicinal dihydroxyl and carboxyl groups is selected from gluconic acid.

[0027] Preferably, to achieve different purposes, those skilled in the art can make conventional adjustments to the amount of the compound having both ortho-dihydroxyl and carboxyl groups and the boron nanosheets. In some embodiments, the molar ratio of the compound having both ortho-dihydroxyl and carboxyl groups to the boron nanosheets having hydroxyl groups is 1:1-3.

[0028] Preferably, the catalyst includes a buffer and a coupling agent. Specifically, those skilled in the art can use the buffer and coupling agent conventionally used in the art for amidation reaction. More specifically, the buffer includes but is not limited to 2-morpholineethanesulfonic acid; the coupling agent includes but is not limited to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0029] Preferably, the concentration of the buffer is 5-10 mM. Preferably, the molar mass ratio of the coupling agent to the compound having both vicinal dihydroxyl and carboxyl groups is 5-20:1.

[0030] Specifically, the polyamide-amine dendrimer is formed by Michael addition reaction of ethylenediamine and methyl acrylate, has a repeating unit of -CH2-CH2-CONHCH2CH2N-, and is an amino-terminated branched macromolecule. More specifically, the molecular weight of the polyamide-amine dendrimer material is 1000-10000; more specifically, the molecular weight of the polyamide-amine dendrimer material is 1000-2000; more specifically, the molecular weight of the polyamide-amine dendrimer material is 1300-1500.

[0031] Preferably, to achieve different purposes, those skilled in the art can make conventional adjustments to the amounts of cationic polymers with amino groups and boron nanosheets with hydroxy groups. Specifically, the mass ratio of cationic polymers with amino groups to boron nanosheets with hydroxy groups is 10-100:1; more specifically, the mass ratio is 40-60:1.

[0032] Preferably, the time for the mixed reaction of the boron nanosheets and the compound having both ortho-dihydroxyl and carboxyl groups is 18-36 hours; and the time for the amidation reaction is 18-36 hours.

[0033] Preferably, the cationic polymer with amino groups is modified on the boron nanosheets and then dialyzed, and the molecular weight of the dialyzed polymer is 2000-20000. Preferably, the molecular weight of the dialyzed polymer is 8000-12000.

[0034] In some embodiments, the specific operation of step S1 is: mixing the boron nanosheets with hydroxyl groups and the cationic polymers with ortho-dihydroxyl groups, and modifying the cationic polymers with ortho-dihydroxyl groups on the boron nanosheets with hydroxyl groups through borate esterification reaction.

[0035] Preferably, the mass ratio of the cationic polymer with ortho-dihydroxyl groups to the boron nanosheets with hydroxyl groups is 1-10:1.

[0036] Preferably, the molar proportion of amino functional groups in the hyperbranched amino polyglycerol is 1-100%. Further preferably, the molar proportion of amino functional groups in the hyperbranched amino polyglycerol is 5-75%. Further preferably, the molar proportion of amino functional groups in the hyperbranched amino polyglycerol is 50-75%.

[0037] Preferably, the molecular weight of the hyperbranched aminopolyglycerol is 2000-20000. Further preferably, the molecular weight is 4000-6000. Most preferably, the molecular weight is 5000.

[0038] Preferably, in step S2, the dialysis time is 24-48h. Preferably, in step S2, the dialysis molecular weight is 2000-20000. Preferably, in step S2, the dialysis molecular weight is 8000-12000.

[0039] Furthermore, the present invention seeks to protect a method for preparing a cationic nanomaterial and a cationic nanomaterial prepared thereby.

[0040] Furthermore, the present invention seeks to protect the use of cationic nanomaterials in the preparation of drugs for treating respiratory inflammation or other inflammatory diseases.

[0041] Preferably, treating respiratory inflammation or other inflammatory diseases refers to: clearing excess cfDNA, NETs, ​​EETs, ROS and / or RNS.

[0042] Preferably, other inflammatory diseases include other inflammatory diseases caused by cfDNA. Other inflammatory diseases caused by cfDNA include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), acute kidney injury (AKI), inflammatory bowel disease (IBD), and periodontitis. In this embodiment, respiratory inflammation includes acute rhinosinusitis, chronic rhinosinusitis, allergic rhinitis, radiation rhinitis, asthma, chronic obstructive pulmonary disease, and acute lung injury.

[0043] Furthermore, the present invention also claims protection for a medicine comprising a therapeutically effective amount of the above cationic nanomaterial.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The cationic nanomaterial prepared by the present invention has good biocompatibility, can inhibit the generation of NETs / EETs by adsorbing cfDNA, and has both antibacterial and antioxidant abilities, and has multiple inflammatory signal targeting effects. In addition, cationic nanomaterials of suitable sizes can be better enriched in the inflammatory site, prolong the in vivo retention time of the material, and can better exert anti-inflammatory effects. The cationic nanomaterial, as a multifunctional nano-platform, can be applied to the treatment of respiratory inflammatory and other inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the synthesis of different cationic nanomaterials. Figure 1 A in the figure is PG1-modified boron nanosheet (BP(BP S , BP M , BP L ))'s synthesis flow chart; Figure 1 B in the figure is hPGA-modified boron nanosheets (B-hPGA S , B-hPGA M , B-hPGA L ))'s synthesis flow chart.

[0047] Figure 2 Fourier transform infrared spectroscopy (FTIR) analysis of B-NS, PG1, gluconic acid-modified B-NS and BP.

[0048] Figure 3 X-ray photoelectron spectroscopy (XPS) analysis diagram of B-NS and BP.

[0049] Figure 4 BP S , BP M , BP L Dynamic light scattering and Zeta potential. Figure 4 A in the formula is BP S , BP M , BP L Dynamic light scattering; Figure 4 The B in it stands for BP S , BP M , BP L Zeta potential.

[0050] Figure 5 BP S , BP M , BP L Transmission electron microscopy image, scale bar: 1 μm.

[0051] Figure 6 B-hPGA M Dynamic light scattering and Zeta potential. Figure 6 A in the equation is B-hPGA M Dynamic light scattering; Figure 6 The B in the formula is B-hPGA M Zeta potential.

[0052] Figure 7 BP S , BP M , BP L Protein adsorption capacity test.

[0053] Figure 8 For B-NS, PG1, BP S , BP M , BP L Cell viability of BEAS-2B cells after 48 h of treatment.

[0054] Fig. 9 PG1, BP S , BP M , BP L , B-hPGA (5:1) and B-hPGA (10:1) were used to test the adsorption capacity of cfDNA. Fig. 9 A and B in the figure are the adsorption efficiency of cfDNA of each material in ddH2O and ddH2O containing 10% FBS, respectively; Fig. 9 C in the figure is the adsorption efficiency of cfDNA by B-hPGA in ddH2O at two polymer ratios.

[0055] Fig.10For B-NS and BP M RONS scavenging ability detection. Fig.10 A, B, and C are the removal efficiencies of DPPH·, ABTS+, and ·OH, respectively.

[0056] Fig.11 For B-NS, PG1 and BP M Antibacterial effect detection. Fig.11 A in the figure is the concentration of BP at different M For E.coli, A + E.coli, K + Antibacterial capacity testing of E.coli and S.aureus; Fig.11 B in the figure is A after treatment with different drugs + E. coli bacterial concentration; Fig.11 C in the figure is A after treatment with different drugs + Growth of E. coli on LB agar plates; Fig.11 D in the figure is A after treatment with different drugs + Live / dead (green / red) staining images of E. coli. Scale bar: 50 μm.

[0057] Fig.12 For B-NS, PG1 and BP M The therapeutic effect on mouse airway inflammation. Fig.12 A in the figure is the detection of cfDNA level in mouse BALF; Fig.12 B in the figure is the total cell count in mouse BALF; Fig.12 C in the figure is a representative image of H&E staining of mouse lungs, scale bar: 50 μm; Fig.12 D in the figure is a representative image of PAS staining in mouse lungs, scale bar: 50 μm; Fig.12 The E in is based on Fig.12 C Scoring of lung inflammation in mice. Fig.12 The F in is based on Fig.12 D. Scoring of mucus production in mouse lungs; Fig.12 G in the figure represents the mRNA expression level of mouse lung inflammatory factors (IL-4, IL-5, IL-13, IL-6, IL-17A, Muc5ac).

[0058] Fig.13 For B-NS, PG1 and BP M Effects on the redox level of mouse lungs. Fig.13 A, B, and C in the figure are the quantification of MDA, SOD, and GSH in mouse BALF, respectively.

[0059] Fig.14 BPM Effects on neutrophils and NETs in mouse lungs. Fig.14 A and B are representative images of Ly6G immunofluorescence staining and CitH3 immunofluorescence staining of mouse lungs, respectively. Scale bar: 50 μm; Fig.14 The C in is based on Fig.14 A Relative quantification of Ly6G fluorescence intensity in immunofluorescence staining images; Fig.14 The D in is based on Fig.14 B Relative quantification of CitH3 fluorescence intensity in immunofluorescence staining images. DETAILED DESCRIPTION

[0060] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0061] Example 1 Preparation of Boron Nanosheets Modified with Polyamidoamine Dendrimers

[0062] (1) Synthesis of Boron Nanosheets (B-NS):

[0063] Step A1: Add 50 mg of magnesium boride (MgB2) to 30 mL of ddH2O and perform a hydrolysis reaction at room temperature for 120 h. Shake the mixture constantly during the reaction.

[0064] Step A2: Centrifuge the product in step A1 at 1500 rpm for 45 min and take the supernatant; centrifuge the supernatant again and take the supernatant.

[0065] Step A3: The product in step A2 was dialyzed in ddH2O (MWCO=1000) for 24 h, and the liquid inside the dialysis bag was taken to obtain a B-NS solution.

[0066] (2) Synthesis of polyamidoamine dendrimer (PAMAM):

[0067] The specific steps of the synthesis of polyamidoamine dendrimers are as follows:

[0068] Step B1: 0.9 g (14.98 mmol) of ethylenediamine was dissolved in 5 mL of methanol, and a methanol solution of 10.3 g (119.6 mmol) of methyl acrylate was added dropwise.

[0069] Step B2: The reaction mixture in Step B1 was stirred at room temperature for 24 h and then distilled to remove methanol and excess methyl acrylate.

[0070] Step B3: The product of Step B2 was dissolved in 10 mL of methanol and added dropwise to 37 g of ethylenediamine.

[0071] Step B4: The reaction mixture in step B3 is stirred at room temperature for 24 hours, and then the solution is distilled to remove methanol and excess ethylenediamine. The product obtained in this step is PAMAM G0.

[0072] Step B5: Add 2 g of PAMAM G0 (dissolved in 10 mL of methanol) to 5.32 g (61.80 mmol) of methyl acrylate solution (10 mL of methanol was first added to the reaction flask to aid dissolution).

[0073] Step B6: The reaction mixture in step B5 was stirred at room temperature for 24 hours, and then the solution was distilled to remove methanol and excess methyl acrylate. The product obtained in this step is PAMAM G0.5.

[0074] Step B7: PAMAM G0.5 obtained in Step B6 (dissolved in 10 mL of methanol) was added dropwise to 37 g of ethylenediamine.

[0075] Step B8: The reaction mixture in step B7 was stirred at room temperature for 24 h, and then the solution was distilled to remove methanol and excess ethylenediamine. The product obtained in this step was PAMAM G1 (PG1, molecular weight 1429).

[0076] (3) Synthesis of Boron Nanosheets (BP) Modified by Polyamidoamine Dendrimers:

[0077] Step C1: Take 18 mg of B-NS (i.e., 0.4 mmol B(OH)2), add 39.2 mg (0.2 mmol) of gluconic acid thereto, shake and react at room temperature for 24 h to obtain gluconic acid-modified B-NS (B-GA).

[0078] Step C2: Add 5 mM 2-morpholineethanesulfonic acid (MES) and 1 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to the reaction in step C1 and mix well. After reacting for 10 min, add 900 mg PG1 and react at room temperature for 24 h.

[0079] Step C3: The product in step C2 is dialyzed in ddH2O (MWCO=10000) for 48 h to remove MES, EDC, and gluconic acid and PG1 not connected to B-NS, and the liquid inside the dialysis bag is taken to obtain boron nanosheets (BP) modified with polyamide-amine dendrimer materials.

[0080] (4) Boron nanosheets (BPs) modified with polyamidoamine dendrimers of different sizes S , BPM , BP L ) synthesis:

[0081] Step D1: Based on step C2, after the reaction is completed, the reaction solution is subjected to ultrasound, the ultrasound power is 500W, and the ultrasound on / off cycle is 5s / 5s. S , the total ultrasound time can be set to 30min; to obtain medium-sized BP M , the total ultrasound time can be set to 15min; to obtain large-size BP L , the total ultrasonic time can be set to 5min.

[0082] Step D2: The product of step D1 was dialyzed in ddH2O (MWCO=10000) for 48 h, and the liquid inside the dialysis bag was taken to finally obtain cationic nanomaterials of different sizes, namely, boron nanosheets (BP S , BP M , BP L ).

[0083] Example 2 Preparation of hyperbranched aminopolyglycerol modified boron nanosheets

[0084] The difference between this embodiment and embodiment 1 is that in this embodiment, since aminopolyglycerol itself has a 1,2-dihydroxy structure, it can be directly modified on the surface of B-NS by boron esterification reaction. The specific operation is as follows:

[0085] (1) Synthesis of Boron Nanosheets (B-NS):

[0086] Step A: consistent with the first embodiment.

[0087] (2) Synthesis of aminopolyglycerol (hPGA):

[0088] Here, the synthesis of 50% amino-modified hPGA is taken as an example.

[0089] Step B1: Hyperbranched polyglycerol (hPG) was prepared by anionic ring-opening polymerization. 1,1,1-tri(hydroxymethyl)propane was deprotonated using potassium methoxide solution under nitrogen protection. Glycidol was slowly added to the reaction vessel at 95°C, and the molecular weight of the hyperbranched polyglycerol (hPG) was controlled according to the monomer / initiator molar ratio. The reaction product was dialyzed in ddH2O and hPG (molecular weight 5000) was obtained by freeze drying.

[0090] Step B2: Under nitrogen protection, hPG (1 g, 0.077 mmol) was dissolved in DMF (20 mL), and excess triethylamine (2.82 mL) was added at room temperature, followed by methanesulfonyl chloride (0.52 ml, 6.72 mol) and the mixture was reacted for 24 h.

[0091] Step B3: The reaction product was centrifuged, the supernatant was taken for rotary evaporation, and the rotary evaporation product was dissolved in 60 mL of DMF. Sodium azide (1.76 g) was added thereto at room temperature, and then stirred at 80° C. for 24 h.

[0092] Step B4: After the reaction system is cooled to room temperature, centrifuge at 8000 g for 10 min, remove the precipitate and take the supernatant.

[0093] Step B5: Add excess ddH2O (2 mL) to the supernatant of Step B4, and then add excess triphenylphosphine (3.6 g), and react at room temperature for 24 h to reduce the azidoketone to the amino group.

[0094] Step B6: After rotary evaporation, the product was dissolved in water, centrifuged, the precipitate was removed, the supernatant was taken, and dialyzed in ddH2O (MWCO=1000) for 48 hours, and the liquid inside the dialysis bag was taken to obtain 50% amino-modified hPGA.

[0095] (3) Boron nanosheets modified with amino polyglycerol of different sizes (B-hPGA S , B-hPGA M , B-hPGA L ) synthesis:

[0096] Step C1: hPGA and B-NS were mixed at room temperature with a mass ratio of hPGA to B-NS of 10:1, and reacted for 24 hours.

[0097] Step C2: Based on step C1, after the reaction is completed, the reaction solution is subjected to ultrasound, the ultrasound power is 500W, and the ultrasound on / off cycle is 5s / 5s. S , the total ultrasound time can be set to 30 min; to obtain medium-sized B-hPGA M , the total ultrasound time can be set to 15min; in order to obtain large-sized B-hPGA L , the total ultrasonic time can be set to 5min.

[0098] Step C3: The product of step C2 was dialyzed in ddH2O (MWCO=10000) for 48 h to finally obtain cationic nanomaterials of different sizes, namely hPGA-modified boron nanosheets (B-hPGA S , B-hPGA M , B-hPGA L ).

[0099] Test Example 1 In vitro performance test of boron nanosheets modified with cationic materials

[0100] 1. Test methods

[0101] (1) Protein adsorption detection: A series of concentrations of cationic nanomaterials were mixed with bovine serum albumin (BSA-FITC) labeled with the fluorescent dye FITC, and the concentration of BSA-FITC was 100 μg / mL. The mixed solution was incubated at 37°C for 30 min and then centrifuged at 11,000 rpm for 10 min. Subsequently, the supernatant was collected and its fluorescence intensity was detected. The protein adsorption ratio (PA) was calculated as follows: PA = (C1-C2) / C1×100%. C1 represents the initial concentration of BSA, and C2 represents the concentration of BSA in the supernatant after centrifugation.

[0102] (2) cfDNA concentration detection: In a black 96-well plate, a series of concentrations of nanomaterials were added to a test solution (ddH2O or ddH2O containing 10% FBS) containing cfDNA (1 μg / mL), and then the Quant-iT TM PicoGreen TM The kit detects cfDNA concentration. According to the instructions, dilute Picogreen fluorescent dye 1000 times with ddH2O and add 50 μL to each well. After mixing, the black 96-well plate was incubated at 37°C in the dark for 15 minutes. The cfDNA concentration can be calculated based on the fluorescence intensity (Ex = 480nm; Em = 520nm) by using an enzyme reader, and the cfDNA removal ability of the functionalized nanosheet can be further calculated.

[0103] (3) Cytotoxicity detection: The cytotoxicity of cationic nanomaterials was evaluated by CCK-8 assay. BEAS-2B cells were inoculated in a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2. When the cell density reached about 50%, the culture medium containing different concentrations of the above cationic nanomaterials was used to continue the culture. After the cells were incubated with the materials for 48 hours, CCK8 reagent was added, and the absorbance was read at 450nm by a microplate reader to calculate the cell activity.

[0104] (4) RONS scavenging ability test

[0105] When testing the DPPH clearance rate, 0.1mM DPPH solution was prepared with anhydrous ethanol, 1mL of cationic nanomaterials with different concentrations and 1mL of DPPH solution were taken and shaken at room temperature for 30min, centrifuged at 5000rpm for 10min, and the absorbance value of the supernatant was measured at 517nm. The DPPH mixed with an equal volume of ddH2O was used as the control group. DPPH clearance rate (%) = (A 对照 -A 样品 ) / A 对照 ×100%.

[0106] Detection of ABTS +When measuring the clearance rate, mix 7.4mM ABTS and 2.6mM K2S2O8 in equal proportions and react at room temperature in the dark for 12 hours. Dilute the mixed solution with anhydrous ethanol until the absorbance at 734nm is about 0.7. This solution is the ABTS working solution. Then take 0.8mL ABTS working solution and 0.2mL cationic nanomaterials of different concentrations and gently shake and mix at room temperature for 6 minutes. Measure the absorbance value at 734nm. Take the ABTS working solution mixed with an equal volume of ddH2O as the control group. ABTS + · Clearance rate (%) = (A 对照 -A 样品 ) / A 对照 ×100%.

[0107] When testing the OH clearance rate, first prepare 9mM salicylic acid solution, 9mM FeSO4 solution and 8.8mM H2O2 solution. Then add 30μL salicylic acid, 30μL FeSO4, 30μL H2O2 and different concentrations of cationic nanomaterials to the EP tube, and make up to 450μL with ddH2O. At the same time, set up a blank group without H2O2 and a control group without cationic nanomaterials, both of which are made up to 450μL with ddH2O. Place the reaction system in a 37℃ water bath for 15min, and measure the absorbance at 510nm. OH clearance rate (%) = A 对照 -(A 样品 -A 空白 ) / A 对照 ×100%.

[0108] (5) Antibacterial ability test

[0109] Escherichia coli (E. coli), ampicillin-resistant Escherichia coli (A + E. coli) and kanamycin-resistant E. coli (K + E.coli) was cultured in Luria-Bertani (LB) medium or LB agar, and Staphylococcus aureus (S.aureus) was cultured in nutrient broth (NB) medium or nutrient agar (NA). Single colonies of each strain were selected for amplification, then diluted and shaken with different concentrations of cationic nanomaterials for 24 hours, and the absorbance at 600nm was measured by Nanodrop to quantify the bacterial concentration. At the same time, the bacterial suspension and different concentrations of cationic nanomaterials were spread on agar plates and cultured in a 37°C incubator. The agar plates were photographed after 24 hours. For bacterial live / dead staining, the bacteria were incubated with 100 μg / mL of the material for 24 hours and then used The BacLightTM Bacterial Viability Kit was used for testing, in which the bacteria were concentrated by centrifugation and incubated with fluorescent dyes (SYTO 9 and propidium), and then 5 μL of the stained bacterial suspension was dropped onto a glass slide and observed using a laser confocal microscope.

[0110] 2. Test results

[0111] Figures 1 to 11 The experimental results of test case 1 are shown.

[0112] Figure 1 Schematic diagram of the synthesis of boron nanosheets modified with cationic materials. Figure 1 A in the figure is PG1-modified boron nanosheet (BP(BP S , BP M , BP L ))'s synthesis flow chart. Figure 1 B in the figure is hPGA-modified boron nanosheets (B-hPGA S , B-hPGA M , B-hPGA L ))'s synthesis flow chart.

[0113] Figure 2 Fourier transform infrared spectroscopy (FTIR) analysis of B-NS, PG1, B-GA and BP. Figure 2 Medium 3411cm -1 The absorption peak at 1072 cm -1 The absorption peak of proved that the modification of gluconic acid was successful, and the characteristic peaks of C=O and NH showed that PG1 was successfully modified onto the surface of B-NS.

[0114] Figure 3 Figure 2 is the X-ray photoelectron spectroscopy (XPS) analysis of B-NS and BP. In the XPS graph, compared with B-NS, the HO-B-OH of BP is reduced, and the characteristic bonds of PG1, CN, C=O and NC, appear.

[0115] Figure 4 BP S , BP M , BP L Dynamic light scattering (DLS) and zeta potential. Figure 4 The A in the figure shows BP S The sizes of BP are mainly distributed in 80-100nm, M The size of BP is mainly distributed in 200-300nm. L The size of the particles is mainly distributed in the range of 500-700nm. Figure 4 The B in the figure shows BP S , BPM , BP L The Zeta potentials were +28.9±4.3mV, +26.6±4.3mV, and +19.4±0.3mV, respectively.

[0116] Figure 5 BP S , BP M , BP L Transmission electron microscopy image of . Scale bar: 1 μm.

[0117] Figure 6 B-hPGA M Dynamic light scattering and Zeta potential. A series of B-hPGA were synthesized by adjusting the mass ratio of hPGA to B-NS at the medium size. M . Figure 6 A in the equation is B-hPGA M Dynamic light scattering. Figure 6 The B in the formula is B-hPGA M Zeta potential.

[0118] Figure 7 BP S , BP M , BP L The protein adsorption capacity of BP S , BP M , BP L The protein adsorption was less than 7%.

[0119] Figure 8 For B-NS, PG1, BP S , BP M , BP L At high concentrations, B-NS, BP S , BP M , BP L Still showed excellent biosafety for BEAS-2B.

[0120] Fig. 9 PG1, BP S , BP M , BP L , B-hPGA (5:1) and B-hPGA (10:1) were used to test the adsorption capacity of cfDNA. Fig. 9 A and B in the figure are PG1, BP in ddH2O and ddH2O containing 10% FBS, respectively. S , BP M , BP L Adsorption efficiency of cfDNA; Fig. 9C in the figure is the adsorption efficiency of B-hPGA on cfDNA at two polymer ratios.

[0121] Fig.10 For B-NS and BP M RONS scavenging ability detection. Fig.10 A in the figure is the detection of DPPH scavenging efficiency. Fig.10 The B in the code stands for ABTS. + ·Detection of removal efficiency. Fig.10 The C in the figure is the detection of OH removal efficiency.

[0122] Fig.11 For B-NS, PG1, BP M Antibacterial effect testing. Fig.11 A in the figure is the concentration of BP at different M For E.coli, A + E.coli, K + Antibacterial ability detection of E.coli and S.aureus. Fig.11 B in the figure is A after treatment with different drugs + E.coli bacterial solution concentration. Fig.11 C in the figure is A after treatment with different drugs + Growth of E. coli on LB agar plates. Fig.11 D in the figure is A after treatment with different drugs + Representative images of live / dead (green / red) staining of E. coli. Scale bar: 50 μm.

[0123] comprehensive Figures 1 to 11 The results show that cationic nanomaterials of different sizes can be prepared by the preparation method of the present invention. FTIR and XPS confirm that BP S , BP M , BP L The material was successfully prepared. Dynamic light scattering results showed that BP S The sizes of BP are mainly distributed in 80-100nm, M The size of BP is mainly distributed in 200-300nm. L The sizes of the BPs were mainly distributed in the range of 500-700 nm, and their Zeta potentials were +28.9±4.3mV, +26.6±4.3mV, and +19.4±0.3mV, respectively. Transmission electron microscopy (TEM) further confirmed that BP S , BP M , BP L The results of Zeta potential also suggest that the charge of the final synthesized material can be controlled by adjusting the mass ratio of cationic polymer to B-NS during the synthesis process.

[0124] BP S , BP M , BP L Both have the characteristics of low protein adsorption and high biosafety, especially at high concentrations. Compared with PG1 alone, BP S , BP M , BP L It still shows excellent biocompatibility, which ensures the safe application of this nanoplatform in vivo.

[0125] In the cfDNA adsorption test, BP M showed better cfDNA adsorption capacity. This may be due to BP M BP S The larger size enables better adsorption and encapsulation of cfDNA. M Shows better than BP L Higher zeta potential leads to stronger electrostatic interaction with negatively charged cfDNA. This result shows that the size and potential of nanomaterials will have a comprehensive impact on their cfDNA adsorption capacity. Based on the above, BP was used in subsequent experiments. M Further research is needed.

[0126] In the RONS clearance experiment, BP M The redox ability of B-NS was similar to that of B-NS. Since B-NS can bind to key components of bacteria (such as lipopolysaccharide or peptidoglycan), B-NS can exhibit antibacterial effects, but whether this effect is affected by polymer coating is still unclear. Fig.11 Display, BP M It still retains good antibacterial effects and is also effective against drug-resistant bacteria. At 100 μg / mL, it can achieve an effect similar to that of antibiotics. In addition, compared with B-NS, BP M showed higher antibacterial ability. At a concentration of 100 μg / mL, BP M The concentration of bacterial culture after treatment was 0.023±0.006IU, while the concentration of bacterial culture after B-NS treatment was 0.133±0.006IU. The significant difference between the two groups was P<0.0001, which may be attributed to BP M The positive charge of the molecule promotes electrostatic binding to bacteria.

[0127] In summary, BP MIt exhibits strong cfDNA adsorption, RONS clearance and bacterial killing capabilities. This invention is the first to prepare a multifunctional nano-platform that integrates cfDNA adsorption, RONS clearance and bacterial killing, and has good clinical transformation potential.

[0128] Test Example 2B-P M Application in mouse models of airway inflammation

[0129] 1. Test method:

[0130] (1) Construction of an ovalbumin (OVA)-induced airway inflammation mouse model: C67BL / 6J mice were used in this experiment. 20 μg of OVA and 75 μL of Freund's complete adjuvant (CFA) were mixed and supplemented to 100 μL with normal saline, which was used as the sensitizing drug for each mouse. On days 0 and 7, each mouse was intraperitoneally injected with 100 μL of the sensitizing drug. After sensitization, on days 14 to 16, each mouse was stimulated by intranasal instillation of 50 μg of OVA. At the same time, a normal saline group was set as a control group. B-NS, PG1 and BP M The treatment group received 50 μg of B-NS, PG1 and BP intranasally 4 h after each challenge. M Administer medication.

[0131] (2) Cell count and cfDNA concentration in bronchoalveolar lavage fluid (BALF): 0.6 mL of normal saline was perfused into the whole lung of the mouse through endotracheal intubation, divided into 3 times, for a total of 1.8 mL. After centrifugation, Pico-green dye was used to detect cfDNA concentration, and the cell pellet was resuspended in 1 mL of PBS, and the total cell count of BALF was performed using a Bio-Rad automatic cell counter.

[0132] (3) Real-time fluorescence quantitative PCR (qRT-PCR): Total lung RNA was extracted with TRIzol and reverse transcribed into cDNA using PrimeScript RT kit. qRT-PCR was performed using ABIQuantStudio 7Flex and SYBR PreMix Ex Taq to quantify mRNA levels. Data were analyzed by the 2-ΔΔCt method.

[0133] (4) Detection of in vivo redox indices: The levels of reduced glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) in mouse BALF were detected according to the instructions provided by the Solarbio detection kit.

[0134] (5) Histopathological examination and immunofluorescence staining: The right main bronchus was ligated, and the left lung was perfused and fixed with 4% paraformaldehyde for 24 h. The lung tissue was then embedded in paraffin, sectioned, and subjected to hematoxylin-eosin (H&E) staining, periodic acid-Schiff (PAS) staining, and immunofluorescence staining.

[0135] 2. Test results

[0136] Figure 12 to Figure 14 The experimental results of Test Case 2 are shown.

[0137] Fig.12 For B-NS, PG1 and BP M Therapeutic effects on airway inflammation in mice. Fig.12 A in the figure shows the detection of cfDNA levels in mouse BALF. Fig.12 B in the figure is the count of the total number of cells in mouse BALF. Fig.12 C is a representative image of H&E staining of mouse lungs, scale bar: 50 μm. Fig.12 D is a representative image of PAS staining in mouse lungs, scale bar: 50 μm. Fig.12 The E in is based on Fig.12 C Scoring of lung inflammation in mice. Fig.12 The F in is based on Fig.12 D Scoring of mucus production in mouse lungs. Fig.12 G in the figure represents the mRNA expression level of mouse lung inflammatory factors (IL-4, IL-5, IL-13, IL-6, IL-17A, Muc5ac).

[0138] Fig.13 For B-NS, PG1 and BP M Effects on redox levels in mouse lungs. Fig.13 A to C in the figure respectively represent the detection of MDA, SOD and GSH in mouse BALF.

[0139] Fig.14 BP M Effects on neutrophils and NETs in mouse lungs. Fig.14 A and Fig.14 B in the figure are representative images of Ly6G immunofluorescence staining and CitH3 immunofluorescence staining of mouse lungs, respectively. Scale bar: 50 μm. Fig.14 The C in is based on Fig.14 A Relative quantification of Ly6G fluorescence intensity in immunofluorescence staining images. Fig.14 The D in is based on Fig.14 B Relative quantification of CitH3 fluorescence intensity in immunofluorescence staining images.

[0140] comprehensive Figure 12 to Figure 14 The results showed that the airway inflammation model mice received BP M After treatment, the cfDNA level in BALF was significantly reduced, and the total number of cells also decreased, reflecting the weakening of the level of inflammatory cells in the lungs, while B-NS treatment alone could not achieve a similar inflammatory relief effect. The results of H&E staining and qRT-PCR in the lungs of mice further showed that BP M Treatment can reduce the infiltration of inflammatory cells in the lungs of mice and reduce the expression of inflammatory factors. In addition, the results of PAS staining and MUC5AC mRNA quantitative analysis in the lungs of mice suggest that BP M The treatment can also reduce the mucus production in the lungs of mice. Since patients with airway inflammation are often accompanied by increased airway mucus, this is also crucial for the treatment of airway inflammation. This result shows that the BP prepared by the present invention is M It can effectively relieve airway inflammation.

[0141] Due to BP M It exhibited excellent antioxidant capacity in vitro, and we further tested its effects on redox indicators in vivo. Fig.13 Showing BP M Effects in the body. M After treatment, the levels of MDA, SOD, and GSH in the BALF of mice were close to those of the control group mice, which was similar to the effect of B-NS and better than the antioxidant effect of PG1.

[0142] Next, the specific mechanism of the effect of cfDNA removal on the inflammatory environment of the lung was further studied. Since cfDNA can stimulate neutrophils to release NETs, ​​fluorescent staining was performed on mouse lung sections to assess the levels of neutrophils and NETs. The results showed that compared with untreated mice, BP M Treatment with BP significantly reduced the fluorescence intensity of Ly6G and CitH3 in the lungs of inflammatory model mice. This result indicates that BP M It can reduce neutrophil infiltration and NETs production in the lungs.

[0143] In summary, the cationic nanomaterial prepared by the preparation method of the present invention can effectively alleviate airway inflammation in vivo and has good application prospects.

[0144] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A method for preparing a cationic nanomaterial, characterized in that: The following steps are involved: S1. connecting a compound having both ortho-dihydroxyl and carboxyl groups to a boron nanosheet having a hydroxyl group by boroesterification, and then modifying a cationic polymer having an amino group on the boron nanosheet by amidation; or The cationic polymer with ortho-dihydroxyl groups was modified on the boron nanosheets with hydroxyl groups through borylation reaction; S2. The boron nanosheets treated in step S1 are dialyzed to obtain cationic nanomaterials; The cationic polymer with amino groups includes polyamidoamine dendrimers; and the cationic polymer with vicinal dihydroxyl groups includes hyperbranched amino polyglycerol.

2. The preparation method according to claim 1, characterized in that: The size of the cationic nanomaterial is 80-700nm.

3. The preparation method according to claim 1, characterized in that: The zeta potential of the cationic nanomaterial is between +15mV and +35mV.

4. The preparation method according to claim 1, characterized in that: The compound having both vicinal dihydroxyl and carboxyl groups is selected from gluconic acid.

5. The preparation method according to claim 1, characterized in that: The boron nanosheets with hydroxyl groups are obtained by subjecting metal borides to a hydrolysis reaction.

6. The preparation method according to claim 1, characterized in that: The molar ratio of the compound with ortho-dihydroxyl and carboxyl groups to the boron nanosheet with hydroxyl groups is 1:1-3; the mass ratio of the cationic polymer with amino groups to the boron nanosheet with hydroxyl groups is 10-100:

1.

7. The preparation method according to claim 1, characterized in that: The molar proportion of amino functional groups in the hyperbranched amino polyglycerol is 1-100%.

8. The cationic nanomaterial prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the cationic nanomaterial according to claim 8 in the preparation of a drug for treating respiratory inflammation or other inflammatory diseases.

10. The use according to claim 9, characterized in that: Treatment of respiratory inflammation or other inflammatory diseases refers to clearing excess cfDNA, NETs, ​​EETs, ROS and / or RNS at the site of inflammation.

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