A preparation method of nanozyme and anti-Klebsiella pneumoniae organ preservation solution composed of the nanozyme

Abstract

The present invention discloses a method for preparing a nanozyme and an anti-Klebsiella pneumoniae organ preservation solution composed of the nanozyme. The preparation method of the nanozyme comprises the following steps: S1, dispersing Ti3AlC2 powder in a mixed solution of LiF and HCl at a solid-liquid ratio of 1:20 (g / mL), and reacting at a constant temperature of 80°C for 36 hours to generate Ti3C2T x MXene precursor; S2, Ti3C2T x The MXene precursor was mixed with a plant polyphenol solution having a concentration of 0.5 g / L and a pH of 5.0, and subjected to a segmented ultrasonic power treatment for 4 h. The anti-Klebsiella pneumoniae organ preservation solution of the present invention was added with Ti3C2T x MXene nanozymes, due to Ti3C2T x The rich oxygen-containing functional groups on the surface of MXene nanozymes can interact with high-viscosity substances such as hydroxyethyl cellulose, successfully reducing viscosity and improving antioxidant effects, significantly prolonging the preservation time of mouse livers, and effectively inhibiting Klebsiella pneumoniae infection, providing better support for organ transplantation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering and organ transplantation technology, and specifically refers to a method for preparing a nanozyme and an anti-Klebsiella pneumoniae organ preservation solution composed of the nanozyme. Background Art

[0002] Donor-derived infection (DDI) refers to the infection of the organ transplant recipient by pathogens infected by the donor through the donated organ. Currently, my country has over 4 million new cases of end-stage liver disease each year, accounting for half of the global total. Organ donation after death is the primary source of livers for liver transplantation in my country, and UW solution (University of Wisconsin solution) and HTK solution (Histidine-Tryptophan-Ketoglutarate solution) are the primary solutions used to preserve these organs, playing a vital role in the field of organ transplantation. Although the overall success rate and survival rate of liver transplantation have significantly improved with the continuous optimization of surgical techniques and immunosuppressant regimens, advances in medical technology have also extended the survival time of patients with organ failure and the length of ICU stay for donors. At the same time, according to research, the lungs / thoracic cavity are the most common sites of infection, followed by the bloodstream, abdomen / biliary tract, urinary tract, perianal area, and liver. Compared with Gram-positive pathogens, infections in liver transplant recipients are more likely to be caused by Gram-negative pathogens. Bloodstream infections caused by the Gram-negative bacterium Klebsiella pneumoniae occur in 6.9% to 18.4% of liver transplant recipients, with Klebsiella pneumoniae accounting for as much as 37% of bacterial infections. Therefore, to reduce the proportion of Klebsiella pneumoniae in bacterial infections, higher requirements are placed on the preservation performance of organ preservation fluids. However, currently available UW / HTK fluids have drawbacks such as high viscosity, diminished antioxidant capacity, and a short shelf life. These not only significantly increase the risk of DDIs but also have unsatisfactory inhibitory effects on Klebsiella pneumoniae.

[0003] In summary, how to effectively reduce the defects of UW solution / HTK solution, such as excessive viscosity, significant attenuation of antioxidant capacity, and effective inhibition of Klebsiella pneumoniae, is one of the core problems that need to be overcome in the field of liver transplantation. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects of existing UW solution and HTK solution, such as high viscosity, large attenuation of antioxidant capacity and unsatisfactory inhibitory effect on Klebsiella pneumoniae, and to provide a method for preparing nanozyme.

[0005] Another object of the present invention is to provide an anti-Klebsiella pneumoniae organ preservation solution composed of the above-mentioned nanozyme.

[0006] To achieve the above object, the present invention adopts the following technical solution: a method for preparing nanozymes, mainly comprising the following steps:

[0007] S1. Disperse Ti3AlC2 powder in a mixed solution of LiF and HCl at a solid-liquid ratio of 1:20 (g / mL) and react at 80°C for 36h to generate Ti3C2T x MXene precursors;

[0008] S2, Ti3C2T x The MXene precursor was mixed with a plant polyphenol solution with a concentration of 0.5 g / L and a pH of 5.0 and treated with segmented ultrasonic power for 4 h to generate Ti3C2T x Ti3C2T with a MXene precursor sheet size of <50nm, thickness <2nm, and plant polyphenol surface coverage ≥98% x MXene nanozyme aqueous solution;

[0009] S3, the Ti3C2T prepared in step S2 x The MXene nanozyme aqueous solution was sterilized through a 0.22 μm filter and freeze-dried to obtain sterilized nanoscale Ti3C2T x MXene nanozyme powder.

[0010] Furthermore, the Ti3C2T x The specific surface area of ​​MXene nanozyme is 300m² / g, and the DPPH free radical scavenging rate is >98%.

[0011] In the mixed solution of LiF and HCl described in step S1, the LiF is of analytical grade, the concentration of HCl is 37%, and the molar ratio of LiF to HCl is 1:3.

[0012] The plant polyphenols described in step S2 are a composite system of dopamine and tannic acid in a mass ratio of 1:1.

[0013] The present invention provides an anti-Klebsiella pneumoniae organ preservation solution composed of nanozymes, which is composed of substances with the following concentrations:

[0014] Histidine: 2.9-3.9 g / L, L-tryptophan: 3.1-4.2 g / L, α-ketoglutaric acid: 1.1-2.0 g / L, KCl: 9.0-11.6 g / L, MgSO4: 0.4-1.1 g / L, NaHCO3: 0.1-0.7 g / L, CaCl2: 0.04-0.09 g / L, hydroxyethyl starch: 45-55 g / L, raffinose: 10-23 g / L, Ti3C2Tx MXene nanozyme: 0.1~1.0 g / L.

[0015] As a preferred embodiment, the anti-Klebsiella pneumoniae organ preservation solution is composed of substances with the following concentrations:

[0016] Histidine: 3.10 g / L, L-tryptophan: 4.08 g / L, α-ketoglutarate: 1.46 g / L, KCl: 9.32 g / L, MgSO4: 0.60 g / L, NaHCO3: 0.42 g / L, CaCl2: 0.06 g / L, hydroxyethyl starch: 50 g / L, raffinose: 15.13 g / L, Ti3C2T x MXene nanozyme: 0.2 g / L.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The anti-Klebsiella pneumoniae organ preservation solution of the present invention is added with Ti3C2T x MXene nanozymes, due to Ti3C2T x The rich oxygen-containing functional groups on the surface of MXene nanozymes can interact with high-viscosity substances such as hydroxyethyl cellulose, successfully reducing viscosity and improving antioxidant effects, significantly prolonging the preservation time of mouse livers, and effectively inhibiting Klebsiella pneumoniae infection, providing better support for organ transplantation.

[0019] (2) The histidine, L-tryptophan, α-ketoglutaric acid and sodium bicarbonate in the anti-Klebsiella pneumoniae organ preservation solution of the present invention jointly form a quaternary buffer system with a pH value of 7.2±0.1 and a buffer capacity of ≥45 mM / pH at 4°C, which can effectively improve the use effect of the present invention.

[0020] (3) During the preparation of the nanozyme and the preservation solution described in the present invention, the nanozyme and the preservation solution are sterilized to ensure the sterility of the preservation solution and the stability of the nanozyme. At the same time, the polyphenol modification in the present invention enables the MXene nanozyme to have both antioxidant (ROS removal efficiency increased by 2 times) and antibacterial (destroy bacterial cell membrane potential) functions, which can effectively overcome the defect of traditional preservation solutions that require the compounding of multiple additives.

[0021] (4) During the preparation process of the present invention, plant polyphenols and ultrasound work synergistically, so that polyphenol molecules are adsorbed between MXene layers through hydrogen bonds. The ultrasonic cavitation effect (local pressure > 100 MPa) destroys the weak interaction between polyphenols and MXene, and the stripping efficiency is increased to more than 4 times that of traditional single ultrasound treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the Ti3C2T in the present invention x Flowchart for the preparation of MXene nanozymes.

[0023] Figure 2 This is a comparison chart of AST data of the anti-Klebsiella pneumoniae organ preservation solution of the present invention and UW, HTK and normal saline at different times.

[0024] Figure 3 This is a comparison chart of AST data after transplantation of organs preserved with the anti-Klebsiella pneumoniae organ preservation solution of the present invention and those preserved with UW, HTK and physiological saline.

[0025] Figure 4 a is a SEM image of Klebsiella pneumoniae cultured in the preservation solution of the present invention.

[0026] Figure 4 b is the SEM image of the control group.

[0027] Figure 4 c is the SEM image of the traditional UW fluid group.

[0028] Figure 4 d is the SEM image of the traditional HTK fluid group. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Example 1

[0031] The Ti3C2T x The preparation process of MXene nanozymes is as follows Figure 1 As shown, it includes the following steps:

[0032] S1. Disperse Ti3AlC2 powder in a mixed solution of LiF and HCl at a solid-liquid ratio of 1:20 (g / mL) and react at 80°C for 36h to generate Ti3C2T x MXene precursor. The Ti3AlC2 powder has a purity greater than or equal to 99.9%, the LiF has an analytical purity, the HCl concentration is 37%, and the molar ratio of LiF to HCl in the mixed solution of LiF and HCl is 1:3.

[0033] S2, Ti3C2T xThe MXene precursor was mixed with a plant polyphenol solution with a concentration of 0.5 g / L and a pH of 5.0 and treated with segmented ultrasonic power for 4 h to generate Ti3C2T x Ti3C2T with a MXene precursor sheet size of <50nm, thickness <2nm, and plant polyphenol surface coverage ≥98% x MXene nanozyme aqueous solution.

[0034] In this step, plant polyphenols will work synergistically with ultrasound, so that polyphenol molecules are adsorbed between MXene layers through hydrogen bonds, and the ultrasonic cavitation effect (local pressure > 100MPa) will destroy the weak interaction between polyphenols and MXene, thereby increasing the stripping efficiency to more than 4 times that of traditional single ultrasonic treatment. The segmented ultrasonic power treatment is used here for 4 hours, specifically the ultrasonic power of the first hour is 100W, and the ultrasonic power of the last 3 hours is 200W. The segmented ultrasonic power treatment used in this embodiment is a process method that optimizes the material preparation performance by dynamically adjusting the energy input. The Ti3C2T generated by this method x The specific surface area of ​​MXene nanozyme is 300m² / g, and the DPPH free radical scavenging rate is >98%.

[0035] S3, the Ti3C2T prepared in step S2 x The MXene nanozyme aqueous solution was sterilized through a 0.22 μm filter and freeze-dried to obtain sterilized nanoscale Ti3C2T x MXene nanozyme powder.

[0036] Example 2

[0037] This embodiment is a nano-scale Ti3C2T generated according to Example 1. x A new anti-Klebsiella pneumoniae organ preservation solution composed of MXene nanozyme powder. The anti-Klebsiella pneumoniae organ preservation solution is composed of the following substances at the following concentrations: histidine: 2.9-3.9 g / L, L-tryptophan: 3.1-4.2 g / L, α-ketoglutaric acid: 1.1-2.0 g / L, KCl: 9.0-11.6 g / L, MgSO4: 0.4-1.1 g / L, NaHCO3: 0.1-0.7 g / L, CaCl2: 0.04-0.09 g / L, hydroxyethyl starch: 45-55 g / L, raffinose: 10-23 g / L, Ti3C2T x MXene nanozyme: 0.1~1.0 g / L.

[0038] As the best embodiment, the above components and concentrations of the anti-Klebsiella pneumoniae organ preservation solution are:

[0039] Histidine: 3.10 g / L, L-tryptophan: 4.08 g / L, α-ketoglutarate: 1.46 g / L, KCl: 9.32 g / L, MgSO4: 0.60 g / L, NaHCO3: 0.42 g / L, CaCl2: 0.06 g / L, hydroxyethyl starch: 50 g / L, raffinose: 15.13 g / L, Ti3C2T x MXene nanozyme: 0.2 g / L.

[0040] It should be noted that when preparing the above-mentioned anti-Klebsiella pneumoniae organ preservation solution, the above-mentioned Ti3C2T x After the substances other than MXene nanozyme are fully mixed, deionized water needs to be added to the solution to 1 L, and sterilized at 121 ° C for 20 min, and then the sterilized Ti3C2T x MXene nanozyme powder, and the Ti3C2T x The concentration of MXene nanozyme can be adjusted to 0.1-1.0 g / L.

[0041] In order to fully illustrate the practical effect of the anti-Klebsiella pneumoniae organ preservation solution of the present invention, the following four groups of experiments were carried out in this embodiment. For the convenience of the following description, Ti3C2T x MXene nanozyme is abbreviated as MXene.

[0042] Experiment 1: To evaluate the scavenging effect of DPPH free radicals, the applicant conducted the following concentration gradient experiments according to the MXene concentration (g / L). The specific results are shown in Table 1

[0043] .

[0044] Where X represents the storage solution containing the MXene solution (* indicates P < 0.01 compared to the saline group). The above-mentioned test method for evaluating the DPPH radical scavenging effect is based on the stable light absorption properties of the free radical and the pairing reaction of antioxidants with its single electron. The experimental conditions are as follows: First, prepare a 0.08-0.1 mmol / L DPPH solution in anhydrous ethanol (stored in the dark). An appropriate amount of the sample solution to be tested is mixed with the DPPH solution in a volume ratio of 1:3 (e.g., 1.0 mL sample + 3.0 mL DPPH solution). Three control groups are also set up: a DPPH solution without sample (A0), a sample solution without DPPH (A_c), and a solvent blank. The mixture is incubated at room temperature in the dark for 30 minutes to allow the antioxidant to fully neutralize the free radicals. The absorbance of each group is then measured at a wavelength of 517 nm (A_s for the sample group, A0 for the free radical control group, and A_c for the sample background).

[0045] The scavenging rate is calculated as follows: Scavenging rate (%) = [ (A0 - (A_s - A_c)) / A0] × 100. This formula accurately reflects free radical scavenging efficacy by eliminating color interference from the sample itself. Experiments should be repeated three times to ensure parallelism (standard deviation ≤ 3%).

[0046] Experiment 2: Statistical analysis of AST in the organ preservation fluid of isolated mouse liver at different time points in different preservation fluid groups was performed. The specific data are shown in Table 2, and the relevant data graphs are shown in Figure 2 shown

[0047] .

[0048] Here, X represents the preservation solution to which MXene solution is added.

[0049] Experiment 3: Comparison of AST (U / L) damage after transplantation. Detailed data are shown in Table 3. Related data graphs are shown in Table 3. Figure 3 shown

[0050] .

[0051] Here, X represents the preservation solution to which MXene solution was added. Mice in the normal saline group could not survive for more than 4 h after liver transplantation, so no data were available.

[0052] By statistically analyzing the AST in the organ preservation fluid of isolated mouse livers at different time points in different preservation fluid groups, it can be concluded that there are statistical differences in the AST of the organ preservation fluid of the present invention and the control group at different times, and the differences are statistically significant (P<0.05), and the differences are all manifestations of deterioration of liver function.

[0053] Experiment 4: The antibacterial effect of the preservation solution of the present invention on Klebsiella pneumoniae. The experimental results are shown in Table 4, and the specific SEM images are shown in Table 4. Figure 4 shown

[0054]

[0055] The specific experimental process of the above-mentioned experiment on the antibacterial effect of Klebsiella pneumoniae is as follows:

[0056] (1) Inoculate the glycerol bacteria (Klebsiella pneumoniae) on a nutrient agar plate and culture at 37°C for 24 hours.

[0057] (2) Take the revived bacteria, pick out single colonies and inoculate them into appropriate liquid culture medium, culture them overnight at 37℃ and 150r / min, and set aside.

[0058] (3) McFarland turbidimetric method was used to determine the bacterial concentration. The bacterial solution was diluted in a gradient manner using sterile culture medium and the concentration was adjusted to 1×10 6 CFU / mL, bacterial solution is reserved.

[0059] (4) The bacterial suspension was mixed with the control group, the traditional UW solution group, the traditional HTK solution group and the preservation solution of the present invention, and cultured at 37°C for about 16 hours.

[0060] (5) Dilute the bacterial suspension after material treatment by 10 4 Then, 50 μL of bacterial solution was spread on LB agar or YPD agar plate, cultured at 37°C for 24 h, and the number of colonies was counted.

[0061] Through the above experimental data and Figure 4 a. Figure 4 b. Figure 4 c and Figure 4 As shown in the SEM image d, the colony count of the preservation solution of the present invention is significantly lower than that of the UW, HTK, and normal saline groups, and the effect of inhibiting Klebsiella pneumoniae is significant. Figure 4 a is a SEM image of Klebsiella pneumoniae cultured in the preservation solution of the present invention, Figure 4 b is the SEM image of the control group. Figure 4 c is the SEM image of the traditional UW liquid group, Figure 4 d is the SEM image of the traditional HTK fluid group.

[0062] As described above, the present invention can be well implemented.

Claims

1. A method for preparing a nanozyme, characterized in that: The main steps include: S1. Disperse Ti3AlC2 powder in a mixed solution of LiF and HCl at a solid-liquid ratio of 1:20 (g / mL) and react at 80°C for 36h to generate Ti3C2T x MXene precursors; S2, Ti3C2T x The MXene precursor was mixed with a plant polyphenol solution with a concentration of 0.5 g / L and a pH of 5.0 and treated with segmented ultrasonic power for 4 h to generate Ti3C2T x Ti3C2T with a MXene precursor sheet size of <50nm, thickness <2nm, and plant polyphenol surface coverage ≥98% x MXene nanozyme aqueous solution; S3, the Ti3C2T prepared in step S2 x The MXene nanozyme aqueous solution was sterilized through a 0.22 μm filter and freeze-dried to obtain sterilized nanoscale Ti3C2T x MXene nanozyme powder; The Ti3C2T x The specific surface area of ​​MXene nanozyme is 300m 2 / g, and the DPPH free radical scavenging rate is >98%.

2. The method for preparing the nanozyme according to claim 1, wherein: In the mixed solution of LiF and HCl described in step S1, the LiF is of analytical grade, the concentration of HCl is 37%, and the molar ratio of LiF to HCl is 1:

3.

3. The method for preparing the nanozyme according to claim 2, wherein: The plant polyphenols described in step S2 are a composite system of dopamine and tannic acid in a mass ratio of 1:

1.

4. An anti-Klebsiella pneumoniae organ preservation solution composed of nanozymes produced by the method for preparing nanozymes according to any one of claims 1 to 3, characterized in that: Composed of the following substances in the following concentrations: Histidine: 2.9-3.9 g / L, L-tryptophan: 3.1-4.2 g / L, α-ketoglutaric acid: 1.1-2.0 g / L, KCl: 9.0-11.6 g / L, MgSO4: 0.4-1.1 g / L, NaHCO3: 0.1-0.7 g / L, CaCl2: 0.04-0.09 g / L, hydroxyethyl starch: 45-55 g / L, raffinose: 10-23 g / L, Ti3C2T x MXene nanozyme: 0.1-1.0 g / L; Among them, the Ti3C2T x The concentration of MXene nanozyme was configured as follows: first, deionized water was added to the solution to 1 L, and sterilized at 121 ° C for 20 min, and then the sterilized Ti3C2T x MXene nanozyme powder, and the Ti3C2T x The concentration of MXene nanozyme was adjusted to 0.1-1.0 g / L.

5. The anti-Klebsiella pneumoniae organ preservation solution composed of nanozymes according to claim 4, characterized in that: Composed of the following substances in the following concentrations: Histidine: 3.10 g / L, L-tryptophan: 4.08 g / L, α-ketoglutaric acid: 1.46 g / L, KCl: 9.32 g / L, MgSO4: 0.60 g / L, NaHCO3: 0.42 g / L, CaCl2: 0.06 g / L, hydroxyethyl starch: 50 g / L, raffinose: 15.13 g / L, Ti3C2T x MXene nanozyme: 0.2 g / L.

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