Cationic nanozyme and its preparation method and application

Abstract

The present invention relates to a cationic nanozyme and a preparation method and application thereof, belonging to the field of biomedicine technology. The cationic nanozyme CTAB@Fe / CDs provided by the present invention is synthesized by a microwave method using a cationic surfactant hexadecyltrimethylammonium bromide as a precursor. CTAB@Fe / CDs exhibits superior peroxidase catalytic activity triggered by activating a Fenton-like reaction and synergistic antifungal activity of CTAB against Candida albicans strains, with the minimum inhibitory concentration and the minimum bactericidal concentration values ​​being 1.56 μg / mL and 10 μg / mL, respectively. On the one hand, the extracellular polysaccharide is cleaved by a high level of hydroxyl radicals under the catalysis of the POD-like catalytic activity of CTAB@Fe / CDs, and on the other hand, the CTAB@Fe / CDs with positive charges on the surface can bind to the negatively charged cell membrane through electrostatic action, change the permeability of the cell membrane, and can kill not only free fungi but also fungi in the biofilm. CTAB@Fe / CDs has strong antifungal and anti-biofilm activity, and is expected to be used to develop or enhance antifungal therapy to prevent drug-resistant Candida albicans infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a cationic nanozyme and a preparation method and application thereof. Background Art

[0002] Candida albicans ( Candida albicans , C. albicans ) is one of the most common human fungal pathogens, which can cause superficial mucosal and life-threatening systemic infections, especially in immunocompromised individuals, and has a significant impact on people's lives. Commonly used antifungal drugs include azoles, polyenes, and echinocandins, but these antifungal drugs have a narrow antifungal spectrum and high toxicity, which limits their application. In addition, the abuse of these antifungal drugs has led to the emergence of many drug-resistant fungal strains. Therefore, the development of new antifungal drugs for the treatment of highly effective antifungal infections has always been an important research topic.

[0003] Nanozymes, nanomaterials with enzyme-mimicking catalytic properties, have recently emerged as a new type of antifungal therapeutic agent, mainly relying on their unique ability to produce toxic products, which can effectively destroy fungi. For example, nanozymes with peroxidase-like (POD) activity can convert H2O2 into highly toxic reactive oxygen species (ROS), which in turn destroy cell structures and inhibit their growth and reproduction, thereby killing pathogenic microorganisms. It is particularly noteworthy that most special nanozymes have good membrane permeability and biocompatibility for fungi, which can avoid fungal resistance. Although artificial nanozymes have made commendable progress in antibacterial applications, their low binding ability to fungi and low diffusivity of ROS and short lifespan severely limit their fungicidal activity. Summary of the invention

[0004] In order to overcome the problems existing in the background technology, the present invention provides a cationic nanozyme and a preparation method and application thereof. The cationic nanozyme is synthesized by a one-step microwave method using a cationic surfactant cetyltrimethylammonium bromide (CTAB) as a precursor and is named iron-doped cationic nanozyme (CTAB@Fe / CDs). CTAB@Fe / CDs exhibits superior peroxidase (POD) catalytic activity triggered by the activation of a Fenton-like reaction and synergistic antifungal activity of CTAB against Candida albicans strains, with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values ​​of 1.56 μg / mL and 10 μg / mL, respectively. On the one hand, the extracellular polysaccharide (EPS) is cleaved by a high level of hydroxyl radicals (•OH) under the catalysis of the POD-like catalytic activity of CTAB@Fe / CDs. On the other hand, the positively charged CTAB@Fe / CDs on the surface can bind to the negatively charged cell membrane through electrostatic interaction, change the permeability of the cell membrane, and ultimately lead to the death of fungi in the fungal biofilm. CTAB@Fe / CDs possess strong antifungal and antibiofilm activities and are expected to be used to develop or enhance antifungal therapies to prevent drug-resistant Candida albicans infections.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] The method for preparing the cationic nanozyme comprises the following steps:

[0007] (1) Add hexadecyltrimethylammonium bromide (CTAB), FeCl3·6H2O and ethylenediamine to acetic acid solution and ultrasonicate for 20-30 min;

[0008] (2) Transfer the solution from step (1) into a high pressure reactor, place it in a microwave digester and heat it to 170-190°C for 1-2 h;

[0009] (3) After the reaction is completed, it is naturally cooled to room temperature, large particles are removed by microfiltration membrane, and then centrifuged at high speed;

[0010] (4) The supernatant obtained by centrifugation is vacuum dried to obtain iron-doped cationic nanozyme CTAB@Fe / CDs.

[0011] Furthermore, the mass ratio of hexadecyltrimethylammonium bromide to FeCl3·6H2O is 0.3-1:1.

[0012] Furthermore, the concentration of the acetic acid solution is 5%; the microfiltration membrane used is a 0.22 μm filter membrane.

[0013] Iron-doped cationic nanozyme CTAB@Fe / CDs was prepared using the above-mentioned preparation method.

[0014] The iron-doped cationic nanozyme CTAB@Fe / CDs is used as an antifungal drug ingredient.

[0015] Furthermore, the fungus is Candida albicans.

[0016] Furthermore, the application is specifically performed by mixing the iron-doped cationic nanozyme CTAB@Fe / CDs with peroxide, and then using it as a sterilizing drug after ultrasonic treatment for 5-10 minutes; wherein the mass ratio of the iron-doped cationic nanozyme CTAB@Fe / CDs to the peroxide is 1:1.5.

[0017] Furthermore, the peroxide is any one of hydrogen peroxide, sodium persulfate or ammonium persulfate.

[0018] Beneficial effects of the present invention:

[0019] The cationic nanozyme of the present invention uses cationic surfactant cetyltrimethylammonium bromide (CTAB) as a precursor and is synthesized by a one-step microwave method. The obtained cationic nanozyme (CTAB@Fe / CDs) has peroxidase catalytic activity and catalyzes H2O2 to produce hydroxyl radicals (·OH). On the other hand, the positively charged surface of the nanozyme effectively combines with the negatively charged fungal cell membrane, thereby increasing its affinity, increasing the diffusivity of ROS, and changing the permeability of the cell membrane. It can kill not only free fungi but also fungi in the biofilm.

[0020] The cationic nanozyme CTAB@Fe / CDs of the present invention exhibits superior peroxidase (POD) catalytic activity triggered by the activation of a Fenton-like reaction and synergistic antifungal activity of CTAB against Candida albicans strains, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MFC) values ​​of 1.56 μg / mL and 10 μg / mL, respectively, and its antibacterial effect is much stronger than that of the positive control drug fluconazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a scanning electron microscope image of the CTAB@Fe / CDs nanozyme of Example 1 of the present invention;

[0022] Figure 2 is the XRD pattern of CTAB@Fe / CDs of Example 1 of the present invention;

[0023] Figure 3 Zeta potential of CTAB@Fe / CDs of Example 1 of the present invention and CTAB@CDs of Comparative Example 1;

[0024] Figure 4The UV-vis absorption spectra of the CTAB@Fe / CDs of Example 1 of the present invention and the CTAB@CDs of Comparative Example 1 catalyzing the oxidation of the peroxidase chromogenic substrate ABTS and H2O2;

[0025] Figure 5 This is the Michaelis-Menten diagram fitting of ABTS and H2O2 catalyzed by CTAB@Fe / CDs in Example 1 of the present invention;

[0026] Figure 6 This is the Michaelis-Menten diagram fitting of ABTS and H2O2 catalyzed by CTAB@CDs in Comparative Example 1 of the present invention;

[0027] Figure 7 This is the fluorescence spectrum of TA detecting •OH;

[0028] Figure 8 •OH spectrum for EPR detection;

[0029] Fig. 9 This is the result of CTAB@Fe / CDs nanozyme cytotoxicity experiment;

[0030] Fig.10 After being treated with different nanozymes C. albicans MFC;

[0031] Fig.11 After being treated with different nanozymes C. albicans Agar plate picture;

[0032] Fig.12 After being treated with different nanozymes C. albicans Agar plate pictures and MIC;

[0033] Fig.13 After being treated with different nanozymes C. albicans Crystal violet staining OD value of biofilm;

[0034] Fig.14 This is a microscope picture of CTAB@Fe / CDs inhibiting the transformation of yeast into mycelium in Example 1 of the present invention;

[0035] Fig.15 The samples treated with CTAB@Fe / CDs (Fig. b) and CTAB@Fe / CDs+H2O2 (Fig. c) in Example 1 and the untreated blank (Fig. a) are shown in Fig. C. albicans SEM images of

[0036] Fig.16 After being treated with CTAB@Fe / CDs of Example 1 and CTAB@CDs of Comparative Example 1 C. albicans Comparison of EPS conditions in biofilms. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0038] The method for preparing the cationic nanozyme comprises the following steps:

[0039] (1) Add 0.03-0.05 g of hexadecyltrimethylammonium bromide (CTAB), 0.05-0.10 g of FeCl3·6H2O and 50-100 μL of ethylenediamine to 25-40 mL of 5% acetic acid solution (unless otherwise specified in the present invention, the percentage concentration of acetic acid is the percentage concentration by mass), and perform ultrasonic treatment for 20-30 min;

[0040] (2) Transfer the mixed solution to a polytetrafluoroethylene high-pressure reactor and place it in a microwave digester heated to 170-190°C at 50 W for 1-2 h;

[0041] (3) After the reaction is completed, it is naturally cooled to room temperature, and large particles are removed with a 0.22 μm filter membrane, followed by high-speed centrifugation;

[0042] (4) The supernatant is vacuum dried to obtain iron-doped cationic nanozyme CTAB@Fe / CDs.

[0043] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0044] Example 1

[0045] Preparation of iron-doped cationic nanozymes (CTAB@Fe / CDs)

[0046] 0.03 g hexadecyltrimethylammonium bromide CTAB, 0.05 g FeCl3·6H2O and 50 μL ethylenediamine were added to 30 mL 5% acetic acid solution, ultrasonically treated for 20 min, and the mixed solution was transferred to a polytetrafluoroethylene high-pressure reactor and placed in a microwave digester heated to 180°C for 1 h. After the reaction was completed, it was naturally cooled to room temperature, and large particles were removed with a 0.22 μm filter membrane. After high-speed centrifugation, the supernatant was vacuum dried to obtain iron-doped cationic nanozyme CTAB@Fe / CDs.

[0047] Comparative Example 1

[0048] Except for not adding FeCl3·6H2O, the other steps were the same as those in Example 1 to prepare the nanomaterial CTAB@CDs.

[0049] Zeta potential test was performed on CTAB@Fe / CDs and CTAB@CDs. The results are as follows: Figure 3 As shown, the Zeta of CTAB@Fe / CDs and CTAB@CDs are 11.7 mV and 5.3 mV, respectively, and CTAB@Fe / CDs has a higher positive charge.

[0050] Analysis and comparison of the products of Example 1 and Comparative Example 1:

[0051] The iron-doped cationic nanozyme CTAB@Fe / CDs obtained in Example 1 and the CTAB@CDs nanomaterial obtained in Comparative Example 1 were analyzed or identified as follows.

[0052] (1) TEM and XRD analysis of CTAB@Fe / CDs

[0053] CTAB@Fe / CDs were characterized by TEM and XRD. The results are as follows: Figure 1 and Figure 2 ,pass Figure 1 It can be seen that CTAB@Fe / CDs is a uniformly distributed amorphous structure. Figure 2 It can be seen that the center position of CTAB@Fe / CDs has about 2 θ = 25°, indicating that CTAB@Fe / CDs has a graphite-like structure.

[0054] (2) Determination of peroxidase-like activity:

[0055] 100 μL of 1 mg / mL iron-doped cationic nanozyme CTAB@Fe / CDs or 100 μL of CTAB@CDs, 100 μL of 5 mmol / L ABTS, and 100 μL of 50 mmol / L H2O2 were added to 2 mL of acetic acid-sodium acetate buffer solution at pH 4.0, mixed thoroughly, and incubated at room temperature for 10 min. The absorbance was measured at 416 nm using a UV-visible spectrophotometer. Each sample was measured 3 times and the average value was taken. The results are shown in Figure 2. Figure 4 ,from Figure 4 It can be seen that the iron-doped cationic nanozyme CTAB@Fe / CDs exhibits peroxidase activity.

[0056] (3) Determination of Michaelis-Menten catalytic kinetic parameters:

[0057] By fixing the concentration of H2O2 at 50 mmol / L and adding different concentrations of ABTS, the kinetics of the nanozyme with ABTS as the substrate was measured; or by fixing the concentration of ABTS at 5 mmol / L and adding different concentrations of H2O2. The absorbance (416 nm) was measured at different reaction times (within 300 s). The kinetic parameters ( K m and V max ):

[0058]

[0059] in v is the reaction rate, [ S ] is the concentration of substrate (ABTS or H2O2), K m is the Michaelis constant, V max is the maximum reaction rate.

[0060] like Figure 5 , Figure 6 Table 1, Michaelis constants of CTAB@Fe / CDs and substrates ABTS and H2O2 K m are 0.214mmol / L and 0.052mmol / L respectively, and the reaction rate constants are 4.84×10 -8 and 17.69×10 -8 mol / L·s; CTAB@CDs for substrates ABTS and H2O2 K m The reaction rates were 0.627 mmol / L and 0.529 mmol / L, respectively, and the reaction rate constants were 1.29×10 -8 and 0.55×10 -8 mol / L·s, which indicates that the introduction of Fe greatly increases the affinity and reaction rate of nanozymes with substrates ABTS and H2O2.

[0061] Table 1 Michaelis-Menten catalytic kinetic parameters

[0062]

[0063] (4) Detection of hydroxyl radicals (•OH):

[0064] Terephthalic acid (TA) was used as a probe to detect •OH. TA was oxidized to 2-hydroxyterephthalic acid (TAOH) in the presence of •OH. 100 μL of 1 mg / mL nanozyme CTAB@Fe / CDs, 200 μL of 5 mg / mL TA, and 100 μL of 50 mmol / L H2O2 were added to 2 mL of acetic acid-sodium acetate buffer solution at pH 4.0, mixed thoroughly, and incubated at room temperature for 4 h. The fluorescence was measured at 425 nm under an excitation wavelength of 305 nm to explore its catalytic activity. The results showed (see Figure 7 ), CTAB@Fe / CDs + H2O2 has a strong ability to produce •OH, and the •OH is determined by electron spin resonance spectrometer (EPR), and the results are consistent with the TA probe detection results ( Figure 8 ).

[0065] (5) Cytotoxicity test:

[0066] The CCK-8 cell viability kit was used to detect the cytotoxicity of the nanozyme. The specific experimental steps were as follows: human umbilical vein endothelial cells (HUVECs, Beina Chuanglian Biotechnology Co., Ltd.) were inoculated in a 96-well plate and cultured for 24 h, and then treated with different concentrations of CTAB@Fe / CDs (0, 5, 10, 20, 40, 80, 160 μg / mL) for 8 h. The cells were rinsed with PBS in batches, and CCK-8 solution was added to each well to a concentration of 10%, incubated at 37°C, and the absorbance was measured at 450 nm; CCK-8 analysis ( Fig. 9 ) showed that CTAB@Fe / CDs had no obvious toxicity to cells.

[0067] (6) Nanoenzyme C. albicans test:

[0068] standard C. albicans ATCC 90028 (purchased from China National Center for Microbiological Culture Collection), two clinical cases C. albicans The isolates were obtained from the vaginal fluid of patients in Yunnan Provincial First People's Hospital and used in efficacy experiments. The minimum inhibitory concentration (MIC) was evaluated by standard microdilution method. Three strains with vigorous growth were selected and washed with RPMI 1640 medium and the concentration of the bacterial solution was adjusted to 10 5 CFU / mL. By multiple dilution method, in the presence of ~10 5Different concentrations of H2O2, CTAB@Fe / CDs+H2O2, CTAB@CDs+H2O2 and antifungal drugs (fluconazole) were prepared in RPMI1640 medium with CFU / mL Candida albicans cells; the concentrations of CTAB@Fe / CDs, CTAB@CDs and fluconazole ranged from 1-25 μg / mL, and the concentration of H2O2 was 50 mmol / L. The control group received cells in RPMI 1640 without any compound. The culture plates were incubated in a thermostatic shaker at 37°C and 120 rpm for 24 h, and the growth was monitored by measuring the absorbance at 600 nm using a multimode reader. The lowest concentration of the compound at which no growth was observed was called the MIC.

[0069] The plate count method was used to estimate the minimum bactericidal concentration (MFC). Specifically, the nanozyme and suspension were diluted and evenly spread on PDA solid culture medium and incubated at 37°C for 48 hours. The concentration at which no Candida albicans colonies appeared on the PDA plate was called MFC. The results showed that ( Fig.10 , Fig.11 and Fig.12 ): CTAB@Fe / CDs for standard and clinical C. albicans The MICs of CTAB@CDs were all 1.56 μg / mL, the MICs of CTAB@CDs were 2.08 μg / mL, 3.12 μg / mL, and 3.12 μg / mL, respectively, and the MIC of fluconazole was all 6.25 μg / mL; C. albicans The MICs of CTAB@Fe / CDs were 10 μg / mL, 13 μg / mL and 13 μg / mL, respectively, the MFCs of CTAB@CDs were 30 μg / mL, 35 μg / mL and 33 μg / mL, and the MIC of fluconazole was greater than 65 μg / mL. It can be seen that CTAB@Fe / CDs has the best antibacterial and bactericidal effects, followed by CTAB@CDs and then fluconazole.

[0070] (7) Nanozyme C. albicans Biofilm removal test:

[0071] The standard C. albicans Add to the wells of a 24-well plate and incubate overnight at 37°C. C. albicansThe biofilms were divided into four groups: blank control group, CTAB@Fe / CDs+H2O2 group, CTAB@CDs+H2O2 group and fluconazole group. The concentrations of CTAB@CDs and fluconazole were set to 200 μg / mL, the concentration of CTAB@Fe / CDs was 5-200 μg / mL, and the concentration of H2O2 was 50 mmol / L. After that, the culture medium in the well plate was gently removed and washed with PBS buffer. Add 200 μL of methanol to the washed well plate and wait for it to air dry. After the methanol was air-dried, 200 μL of crystal violet (CV) (0.1 mg / mL) was added to each well plate and incubated for 30 min. After the incubation, wash again with PBS buffer solution to clean the excess dye. Finally, add 200 μL of glacial acetic acid to the well plate to dissolve CV and measure the absorbance value at 590nm using an ELISA plate. The results showed that ( Fig.13 ): When the concentration of CTAB@Fe / CDs was 15 μg / mL, it could effectively destroy the biofilm formed by CV staining, while increasing the concentration of CTAB@CDs and fluconazole to 200 μg / mL did not have a good effect.

[0072] In addition, biofilm formation and fungal pathogenicity are dependent on the transformation of yeast cells into filamentous hyphae. Microscopic observation of Candida albicans cells cultured in the presence of subinhibitory concentrations of CTAB@Fe / CDs is shown in Figure 2. Fig.14 As shown (control is the blank control group), through Fig.14 It can be seen that CTAB@Fe / CDs had a significant effect on hyphal transformation. In the control cells without CTAB@Fe / CDs, cell growth was mainly manifested as hyphae, while at 0.5 MIC, cells grew mainly as yeast cells without hyphae, and an increase in fermentation and hyphal formation was observed at sub-MIC (0.25 ~ 0.0625).

[0073] At the same time, SEM images (such as Fig.15 As shown, Figure a is a blank control, Figure b is treated with CTAB@Fe / CDs, and Figure c is treated with CTAB@Fe / CDs+H2O2) C. albicans Compared with the blank control group, after incubation with nanozymes, C. albicans The cell membrane was broken to some extent and no longer smooth. After being treated with CTAB@Fe / CDs+H2O2, C. albicans The cell membrane was completely ruptured, causing obvious necrosis.

[0074] Like other biofilms, C. albicansBiofilms are usually composed of a thick layer of exopolysaccharides (EPS) and an extracellular matrix (ECM) composed of proteins. This ECM contributes to the thickness and total biomass of the biofilm, making it capable of resisting fungal drugs. Since EPS is an important component of biofilm biomass, biofilm formation is closely related to EPS production. Therefore, we investigated the biofilm growth and bioavailability of CTAB@Fe / CDs in the presence of C. albicans EPS generation ( Fig.16 ). The results showed that compared with the control, CTAB@Fe / CDs+H2O2 significantly reduced the production of EPS (72% EPS reduction compared with the group without any treatment), indicating that CTAB@Fe / CDs can successfully inhibit the synthesis of EPS and prevent the formation of biofilm.

[0075] The above experimental results confirm that the CTAB@Fe / CDs synthesized in the present invention produces a high level of hydroxyl radicals (•OH) due to its positive surface charge and peroxidase-like activity. The positive charge on the surface combines with the negatively charged cell membrane through electrostatic action, changing the permeability of the cell membrane. It can kill not only free fungi but also fungi in biofilms, and can be used as a new type of antifungal agent.

[0076] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a cationic nanozyme, characterized in that: The following steps are involved: (1) Add hexadecyltrimethylammonium bromide (CTAB), FeCl3·6H2O and ethylenediamine to acetic acid solution and ultrasonicate for 20-30 min; (2) Transfer the solution from step (1) into a high pressure reactor, place it in a microwave digester and heat it to 170-190°C for 1-2 hours; (3) After the reaction is completed, it is naturally cooled to room temperature, large particles are removed by microfiltration membrane, and then centrifuged at high speed; (4) The supernatant obtained by centrifugation is vacuum dried to obtain iron-doped cationic nanozyme CTAB@Fe / CDs.

2. The preparation method according to claim 1, characterized in that: The mass ratio of hexadecyltrimethylammonium bromide to FeCl3·6H2O is 0.3-1:

1.

3. The preparation method according to claim 1, characterized in that: The concentration of the acetic acid solution is 5%; the microfiltration membrane used is a 0.22 μm filter membrane.

4. The iron-doped cationic nanozyme CTAB@Fe / CDs prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the iron-doped cationic nanozyme CTAB@Fe / CDs as claimed in claim 4 in the preparation of drugs for killing Candida albicans.

6. The use according to claim 5, characterized in that The iron-doped cationic nanozyme CTAB@Fe / CDs was mixed with peroxide and used as a sterilizing agent after ultrasonic treatment for 5-10 minutes; the weight ratio of iron-doped cationic nanozyme CTAB@Fe / CDs to peroxide was 1:1.

5.

7. The use according to claim 6, characterized in that The peroxide is any one of hydrogen peroxide, sodium persulfate or ammonium persulfate.

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