Preparation method of nano-enzyme and anti-klebsiella pneumoniae organ preserving fluid prepared from nano-enzyme

Abstract

The invention discloses a preparation method of a nano-enzyme and an anti-klebsiella pneumoniae organ preserving fluid formed by the nano-enzyme, the preparation method of the nano-enzyme comprises the following steps: S1, dispersing Ti3AlC2 powder in a mixed solution of LiF and HCl according to a solid-to-liquid ratio of 1: 20 (g / mL), and reacting for 36 hours at a constant temperature of 80 DEG C to generate a Ti3C2TxMXene precursor; and S2, mixing the Ti < 3 > C < 2 > T < x > MXene precursor with a plant polyphenol solution with the concentration of 0.5 g / L and the pH value of 5.0, and carrying out segmented ultrasonic power treatment for 4 hours. The Ti3C2TxMXene nano-enzyme is added into the anti-klebsiella pneumoniae organ preserving fluid, and due to the fact that oxygen-containing functional groups rich on the surface of the Ti3C2TxMXene nano-enzyme can interact with high-viscosity substances such as hydroxyethyl cellulose, the viscosity is successfully reduced, the anti-oxidation effect is improved, the preservation time of mouse livers is remarkably prolonged, and the anti-klebsiella pneumoniae organ preserving fluid has the advantages that the anti-klebsiella pneumoniae organ preserving fluid can be used for preparing anti-klebsiella pneumoniae organs; the Klebsiella pneumoniae infection can be effectively inhibited, and better support is provided for organ transplantation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering and organ transplantation, and specifically refers to a preparation method of nanozyme and an organ preservation solution against Klebsiella pneumoniae composed of the nanozyme. Background Art

[0002] Donor-derived infection (DDI) refers to the infection of the recipient of an organ transplantation caused by the pathogens infected by the donor through the donated organ. At present, more than 4 million new end-stage liver disease patients are added annually in China, accounting for 1 / 2 of the global total. Organ donation after citizen's death is the main source of donor livers for liver transplantation in China, and UW solution (University of Wisconsin solution) and HTK solution (Histidine - Tryptophan - Ketoglutarate solution) are the main solutions used for the preservation of these organs, playing an important role in the field of organ transplantation. Although with the continuous optimization of surgical techniques and immunosuppressive regimens, the overall success rate and survival rate of liver transplantation surgeries have been significantly improved, the progress of medical technology has also extended the survival time of patients with organ failure and the length of stay in the donor ICU. At the same time, according to research findings, the lung / thorax is the most common infection site, followed by the bloodstream, abdomen / biliary tract, urinary tract, perianal area, and liver. Compared with Gram-positive pathogens, the infections of liver transplant recipients are more likely to be caused by Gram-negative pathogens. 6.9% - 18.4% of liver transplant recipients develop bloodstream infections caused by the Gram-negative bacterium Klebsiella pneumoniae, and Klebsiella pneumoniae accounts for as high as 37% of bacterial infections. Therefore, in order to reduce the proportion of Klebsiella pneumoniae in bacterial infections, higher requirements are put forward for the preservation effect of the organ preservation solution. However, the existing UW solution / HTK solution currently has defects such as too high viscosity, attenuation of antioxidant capacity, and short preservation time limit, which will not only significantly increase the risk of DDI, but also have an unsatisfactory inhibitory effect on Klebsiella pneumoniae.

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

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

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

[0006] To achieve the above object, the present invention adopts the following technical solutions: A preparation method of nanozyme, mainly including the following steps: 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 a constant temperature of 80 °C for 36 h to generate Ti3C2T x MXene precursor; S2. Mix the Ti3C2T x MXene precursor with a plant polyphenol solution with a concentration of 0.5 g / L and a pH of 5.0, and perform segmented ultrasonic power treatment for 4 h to generate Ti3C2T x MXene nanozyme aqueous solution with a lamellar size of the Ti3C2T x MXene precursor < 50 nm, thickness < 2 nm, and plant polyphenol surface coverage rate ≥ 98%; S3. Sterilize the Ti3C2T x MXene nanozyme aqueous solution prepared in step S2 through a 0.22 μm filter and perform freeze-drying to obtain sterilized nano-scale Ti3C2T x MXene nanozyme powder.

[0007] Furthermore, the specific surface area of the Ti3C2T x MXene nanozyme is 300 m² / g, and the DPPH free radical scavenging rate > 98%.

[0008] In the mixed solution of LiF and HCl in step S1, the LiF is of analytical pure grade, the concentration of HCl is 37%, and the molar ratio of LiF to HCl is 1:3.

[0009] The plant polyphenol in step S2 is a compound system of dopamine and tannic acid with a mass ratio of 1:1.

[0010] The present invention provides an organ preservation solution against Klebsiella pneumoniae composed of nanozyme, which is 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.

[0011] Preferably, the Klebsiella pneumoniae-resistant organ preservation solution consists of substances with 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: 15.13g / L, Ti3C2T x MXene nanozyme: 0.2 g / L.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Ti3C2T x MXene nanozyme is added to the Klebsiella pneumoniae-resistant organ preservation solution of the present invention. Due to the rich oxygen-containing functional groups on the surface of Ti3C2T x MXene nanozyme, it can interact with high-viscosity substances such as hydroxyethyl cellulose, successfully reducing the viscosity and improving the antioxidant effect, significantly extending the preservation time of mouse liver, and effectively inhibiting Klebsiella pneumoniae infection, providing better support for organ transplantation.

[0013] (2) Histidine, L-tryptophan, α-ketoglutaric acid and sodium bicarbonate in the Klebsiella pneumoniae-resistant 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.

[0014] (3) During the preparation of the nanozyme and the preservation solution of the present invention, the nanozyme and the preservation solution are respectively 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 endows the MXene nanozyme with dual functions of antioxidant (the efficiency of scavenging ROS is increased by 2 times) and antibacterial (destroying the bacterial cell membrane potential), which can effectively overcome the defect that traditional preservation solutions need to be compounded with multiple additives.

[0015] (4) During the preparation process of the present invention, the synergistic effect of plant polyphenols and ultrasound enables polyphenol molecules to be adsorbed between MXene layers through hydrogen bonds. The ultrasonic cavitation effect (local pressure > 100 MPa) destroys the weak interaction between polyphenols and MXene, increasing the stripping efficiency to more than 4 times that of traditional single ultrasonic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the preparation flow chart of Ti3C2T x MXene nanozyme in the present invention.

[0017] Figure 2 This is a comparison chart of AST data of the Klebsiella pneumoniae - resistant organ preservation solution of the present invention, UW, HTK, and normal saline at different times.

[0018] Figure 3 This is a comparison chart of AST data after transplantation of organs preserved with the Klebsiella pneumoniae - resistant organ preservation solution of the present invention, UW, HTK, and normal saline.

[0019] Figure 4 a is the SEM image of Klebsiella pneumoniae cultured with the preservation solution of the present invention.

[0020] Figure 4 b is the SEM image of the control group.

[0021] Figure 4 c is the SEM image of the traditional UW solution group.

[0022] Figure 4 d is the SEM image of the traditional HTK solution group. Detailed implementation manners

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Example 1

[0025] The Ti3C2T x preparation process of MXene nanozyme is as Figure 1 shown, and it includes the following steps: S1. Disperse Ti3AlC2 powder at a solid - liquid ratio of 1:20 (g / mL) in a mixed solution of LiF and HCl, and react at a constant temperature of 80 °C for 36 h to generate Ti3C2T x MXene precursor. Among them, the purity of the Ti3AlC2 powder is greater than or equal to 99.9%, the purity of the LiF is of analytical - pure grade, the concentration of HCl is 37%, and in the mixed solution of LiF and HCl, the molar ratio of LiF to HCl is 1:3.

[0026] S2. Mix the Ti3C2T x MXene precursor with a plant polyphenol solution with a concentration of 0.5 g / L and a pH of 5.0, and perform segmented ultrasonic power treatment for 4 h to generate Ti3C2T xTi3C2T with MXene precursor having a sheet size < 50 nm, a thickness < 2 nm, and a plant polyphenol surface coverage rate ≥ 98% x MXene nanozyme aqueous solution.

[0027] In this step, plant polyphenols and ultrasound act synergistically, enabling polyphenol molecules to adsorb between MXene sheets through hydrogen bonds. The ultrasonic cavitation effect (local pressure > 100 MPa) will disrupt the weak interaction between polyphenols and MXene, thus increasing the exfoliation efficiency to more than 4 times that of traditional single ultrasonic treatment. In this case, segmented ultrasonic power treatment is carried out for 4 h. Specifically, the ultrasonic power in the first 1 h is 100 W, and the ultrasonic power in the next 3 h is 200 W. The segmented ultrasonic power treatment adopted in this example is a process method for optimizing the material preparation performance by dynamically adjusting the energy input. The Ti3C2T generated by this method x The specific surface area of the MXene nanozyme is 300 m² / g, and the DPPH free radical scavenging rate > 98%.

[0028] S3. The Ti3C2T prepared in step S2 x The MXene nanozyme aqueous solution is sterilized through a 0.22 μm filter and freeze-dried to obtain a sterilized nano-scale Ti3C2T x MXene nanozyme powder.

[0029] Example 2

[0030] This example is a brand-new Klebsiella pneumoniae organ preservation solution composed of the nano-scale Ti3C2T x MXene nanozyme powder. The Klebsiella pneumoniae organ preservation solution is composed of the following substances at the following concentrations: specifically, 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.

[0031] As the optimal implementation method, the above components and concentration values of the Klebsiella pneumoniae organ preservation solution are: 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.

[0032] It should be noted that when preparing the above-mentioned Klebsiella pneumoniae organ preservation solution, after fully mixing the substances other than Ti3C2T x MXene nanozyme, it is necessary to first supplement deionized water to 1 L in this solution, sterilize it at 121 °C for 20 min, and then add the aseptically treated Ti3C2T x MXene nanozyme powder, and adjust the concentration of this Ti3C2T x MXene nanozyme to 0.1 - 1.0 g / L.

[0033] To fully illustrate the actual effect of the Klebsiella pneumoniae organ preservation solution of the present invention, the following four groups of experiments were carried out in this example. For the convenience of the following description, hereinafter Ti3C2T x MXene nanozyme is abbreviated as MXene.

[0034] Experiment 1: Evaluation of the scavenging effect on DPPH radicals. The applicant carried out the following concentration gradient experiments according to the MXene concentration (g / L), and the specific results are shown in Table 1 .

[0035] Among them, X represents the preservation solution added with MXene solution (* indicates P < 0.01 compared with the normal saline group). The test method for the above-mentioned DPPH radical scavenging effect evaluation is based on the stable light absorption characteristics of this radical and the single-electron pairing reaction of antioxidants to it. The test conditions are as follows: First, prepare a 0.08 - 0.1 mmol / L DPPH anhydrous ethanol solution (stored in the dark), take an appropriate amount of the sample solution to be tested and mix it with the DPPH solution at a volume ratio of 1:3 (such as 1.0 mL sample + 3.0 mL DPPH solution). At the same time, set three control groups, namely the DPPH solution without the sample (A0), the sample solution without DPPH (A_c), and the solvent blank. Let the mixture stand in the dark at room temperature for 30 minutes to allow the antioxidant to fully neutralize the radicals. Subsequently, measure the absorbance of each group at a wavelength of 517 nm (A_s is the sample group, A0 is the radical control group, and A_c is the sample background).

[0036] The clearance rate calculation formula is: Clearance rate (%) = [(A0 - (A_s - A_c)) / A0] × 100. This formula accurately reflects the free radical scavenging efficiency by eliminating the interference of the sample's own color. The experiment needs to be repeated three times to ensure parallelism (standard deviation ≤ 3%).

[0037] Experiment 2: Statistical analysis was performed on AST in the organ preservation solution of isolated mouse livers at different time points for different preservation solution groups. The specific data are shown in Table 2, and the relevant data graphs are as Figure 2 shown .

[0038] Among them, X represents the preservation solution added with the MXene solution.

[0039] Experiment 3: For the comparison of the degree of AST (U / L) injury after transplantation, the detailed data are shown in Table 3, and the relevant data graphs are as Figure 3 shown .

[0040] Among them, X represents the preservation solution added with the MXene solution. Mice in the normal saline group could not survive more than 4 h after liver transplantation, so there is no data.

[0041] By performing statistical analysis on AST in the organ preservation solution of isolated mouse livers at different time points for different preservation solution groups, it can be concluded that there are statistical differences among multiple groups in the comparison of AST between the organ preservation solution 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 liver function deterioration.

[0042] Experiment 4: The antibacterial effect of the preservation solution of the present invention against Klebsiella pneumoniae. The experimental results are shown in Table 4, and the specific SEM images are as Figure 4 shown

[0043] The specific experimental process of the above experiment on the antibacterial effect against Klebsiella pneumoniae is as follows: (1) Inoculate the glycerol bacteria (Klebsiella pneumoniae) on a nutrient agar plate and culture at 37 °C for 24 h for standby.

[0044] (2) Take the revived bacteria, pick single colonies and inoculate them into appropriate liquid media respectively, and culture overnight at 37 °C and 150 r / min for standby.

[0045] (3) Determine the bacterial concentration by the McFarland turbidity method, and use sterile medium to serially dilute the bacterial solution, and adjust the concentration to 1×10 6 CFU / mL, and the bacterial solution is for standby.

[0046] (4) The bacterial suspensions of bacteria were respectively 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 h.

[0047] (5) After diluting the bacterial suspension after material treatment by 10 4 , 50 μL of the bacterial solution was taken and spread on an LB agar or YPD agar plate, and cultured at 37 °C for 24 h, and the number of colonies was counted.

[0048] From the above experimental data and Figure 4 a, Figure 4 b, Figure 4 c and Figure 4 the SEM images shown in d, it can be seen that the colony count of the preservation solution of the present invention is significantly decreased compared with the colony counts of the UW, HTK, and normal saline groups, and it has a significant effect in inhibiting Klebsiella pneumoniae. Among them, Figure 4 a is the SEM image of Klebsiella pneumoniae cultured with 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 solution group, Figure 4 d is the SEM image of the traditional HTK solution group.

[0049] As described above, the present invention can be well realized.

Claims

1. A preparation method of a nanozyme, characterized in that, It mainly includes the following steps: S1. Disperse Ti3AlC2 powder in a mixed solution of LiF and HCl at a solid-liquid ratio of 1:20 (g / mL), and react for 36 h under the condition of constant temperature at 80 °C to generate a Ti3C2Tx x MXene precursor; S2. Mix the Ti3C2T x MXene precursor with a phlorotannin solution with a concentration of 0.5 g / L and a pH of 5.0, and perform segmented ultrasonic power treatment for 4 h to generate Ti3C2T x MXene nanzyme aqueous solution with a sheet size of <50 nm, a thickness of <2 nm, and a phlorotannin surface coverage rate of ≥98% x MXene nanzyme aqueous solution; S3. Sterilize the Ti3C2T x MXene nanozyme aqueous solution prepared in step S2 through a 0.22 μm filter and freeze-dry it to obtain sterilized nano-scale Ti3C2T x MXene nanozyme powder.

2. The preparation method of the nanozyme according to claim 1, wherein The Ti3C2T x The specific surface area of the MXene nanozyme is 300 m² / g, and the DPPH free radical scavenging rate > 98%.

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

3.

4. The preparation method of the nanozyme according to claim 2, characterized in that, The plant polyphenol described in step S2 is a compound system of dopamine and tannic acid with a mass ratio of 1:

1.

5. An organ preservation solution against Klebsiella pneumoniae composed of nanozymes generated by the preparation method of the nanozyme according to any one of claims 1 to 4, characterized in that, It consists of substances with 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.

6. The Klebsiella pneumoniae organ preservation solution composed of the nanozyme according to claim 5, characterized in that, It consists of substances with 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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