Preparation method and application of a photothermal nanoreagent targeting bacteria

Abstract

This invention discloses a method for preparing and applying a photothermal nanoreagent targeting bacteria. This invention utilizes Ti3C2T... x Using MXene as a matrix, a photothermal nanoreagent MXene@PDA@Peptide (MPP) was constructed by sequentially modifying the surface of Ti3C2Tx MXene with polydopamine and grafting it with the peptide CAEKA, which has the function of recognizing bacteria. Under the recognition of the peptide CAEKA, the photothermal Ti3C2Tx MXene was targeted. x MXene nanosheets are guided to the surface of bacterial cells, achieving effective sterilization at a relatively low overall temperature of around 50°C (or achieving more efficient sterilization at the same overall temperature level, exhibiting superior sterilization performance compared to pure Ti3C2Tx MXene). This makes them particularly suitable for the preparation of therapeutic drugs for subcutaneous abscesses caused by methicillin-resistant Staphylococcus aureus (MRSA) infection.

Description

TECHNICAL FIELD

[0001] The biological nanomaterial of the present application, in particular, relates to a preparation method and application of a bacterium-targeting photothermal nano reagent. BACKGROUND

[0002] It is well known that the discovery of antibiotics is a historic breakthrough in the process of human resistance to bacterial infection, but the abuse of antibiotics and the adaptive evolution of bacteria under the pressure of antibiotics make bacteria resistant, which poses a serious threat to human health. However, the development of new antibiotics has a long cycle, increasing difficulty and shortening effective period, which further aggravates the human health. Therefore, it is urgent to develop a new type of antibacterial material different from antibiotics to cope with the current serious bacterial infection problem.

[0003] In recent years, with the rapid development of nanotechnology, photothermal therapy (PTT) based on nanomaterials has great advantages and potential in the treatment of bacterial infection. Photothermal therapy is an exogenous, non-invasive physical therapy that can effectively kill bacteria without inducing bacterial resistance. Among them, transition metal carbide (MXene) as a new star in two-dimensional nanomaterials has many excellent properties, such as good dispersibility in water, good biocompatibility, good absorption capacity in the near-infrared region and excellent photothermal conversion performance, etc., so it can be used as an ideal photothermal reagent for photothermal therapy of bacterial infection. However, due to the lack of recognition ability of MXene to bacteria, it will inevitably damage healthy biological tissues during treatment, which to some extent limits its application in photothermal therapy.

[0004] Therefore, it is necessary to modify the surface of MXene to make it have the ability to target bacteria (photothermal nano reagent capable of targeting bacteria) to achieve bactericidal effect at a lower overall temperature level. SUMMARY

[0005] In view of the above defects of the prior art, the present application provides a preparation method and application of a photothermal nano reagent capable of targeting bacteria.

[0006] The technical purpose of the present application is achieved by the following technical scheme:

[0007] The present application provides a preparation method of a bacterium-targeting photothermal nano reagent, comprising the following steps:

[0008] S1, using Ti3AlC2 clay as raw material, selectively etching the Al element layer thereof with an etchant, and ultrasonicating under inert gas protection to obtain Ti3C2T x MXene nanosheet;

[0009] S2, under alkaline conditions, Ti3C2T x Surface modification of MXene nanosheets to form a polydopamine coating, centrifugation, washing, collecting the precipitate to obtain MXene@PDA (MP);

[0010] S3, under inert gas protection, a short peptide capable of identifying bacteria is linked to the surface of the MXene@PDA obtained in S2 to obtain MXene@PDA@Peptide.

[0011] Further, the etchant of the present application is HCl / LiF, the concentration of HCl is 8-10 mol / L, and the concentration of LiF is 0.04-0.06 g / mL; preferably, the etching temperature is 40±1℃, and the etching time is 36±0.5h.

[0012] Further, the amount ratio of Ti3AlC2 clay to etchant in S1 of the present application is (1-2) g:(30-40) ml.

[0013] Further, in S2 of the present application, Ti3C2T x The surface modification substance of MXene nanosheets is hydrochloric acid dopamine, and its concentration range is 1-2 mg / mL.

[0014] Further, in the present application, the reaction time of S2 is 12±0.1h; it is carried out in a dark environment; preferably, the pH value of the alkaline aqueous solution in S2-S3 is 8.3-9.0, and the pH value is adjusted by tris(hydroxymethyl) aminomethane or sodium hydroxide.

[0015] Further, the amino acid sequence of the short chain peptide in S3 of the present application is cysteine-alanine-glutamic acid-lysine-alanine; preferably, the concentration of the short chain peptide is 0.5-2.0 mg / mL.

[0016] Further, in S3 of the present application, the short peptide is linked to the MXene@PDA obtained in S2 by Michael addition reaction or Schiff base reaction (stirring at room temperature for 48h) between thiol or amino and polydopamine (PDA), and the mass ratio of MXene@PDA obtained in S2 to the short peptide chain is 1:(1-3).

[0017] Further, the bacteria in S3 of the present application are selected from gram-negative bacteria, gram-positive bacteria and drug-resistant bacteria; preferably, the bacteria are selected from Escherichia coli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0018] Another technical purpose of the present application is to provide an application of the photothermal nano reagent prepared by the above preparation method in antibacterial infection.

[0019] Further, the bacteria according to the present application are resistant to methicillin-resistant Staphylococcus aureus.

[0020] The present application has the following beneficial effects relative to the prior art:

[0021] (1) The Ti3C2T x The targeted photothermal antibacterial strategy of MXene provides a new choice for the synthesis of precise and efficient antibacterial materials.

[0022] (2) The preparation method of the present application uses biomimetic material polydopamine to modify the surface of Ti3C2T x MXene, which has more excellent biocompatibility.

[0023] (3) Compared with non-selective photothermal therapy, the targeted photothermal nanoreagent constructed by the present method can achieve more significant bactericidal effect at the same overall temperature.

[0024] (4) The targeted photothermal nanoreagent obtained by the present application can effectively treat subcutaneous abscess caused by methicillin-resistant Staphylococcus aureus infection. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 (a) is a schematic diagram of the preparation process of the targeted photothermal nanobactericidal reagent; (b) is a schematic diagram of the treatment of bacterial infection in vivo by the targeted photothermal nanoreagent prepared by us and its potential antibacterial mechanism.

[0026] Figure 2 (a)-(b) are transmission electron microscopy (TEM) and atomic force microscopy (AFM) patterns of Ti3C2Tx MXene and MP prepared in Example 1; (c)-(d) are scanning electron microscopy (SEM) patterns of Ti3C2Tx MXene and MP, respectively; (e) is the Tyndall phenomenon pattern of Ti3C2Tx MXene and MP colloidal solution, respectively; (f) is the X-ray diffraction (XRD) pattern of Ti3AlC2 MAX clay, Ti3C2Tx MXene and MP; (g) is the Fourier transform infrared spectroscopy (FTIR) spectrum of polydopamine (PDA), Ti3C2Tx MXene and MP.

[0027] Figure 3 X-ray photoelectron spectroscopy (XPS) spectrum of MPP prepared in Example 1.

[0028] Figure 4 In vitro photothermal antibacterial effect of MPP prepared in Example 1.

[0029] Figure 5 Targeting effect of MPP prepared in Example 1 on methicillin-resistant Staphylococcus aureus.

[0030] Figure 6 In vitro biocompatibility of the MPP prepared for Example 1.

[0031] Figure 7 Therapeutic effect of the MPP prepared for Example 1 on subcutaneous abscess caused by MRSA infection. DETAILED DESCRIPTION

[0032] In order to better illustrate the technical objects, technical schemes and advantages of the present application, the present application will be further described in conjunction with the drawings and specific embodiments.

[0033] The products used in the present application are as follows:

[0034] The short-chain peptide CAEKA is prepared by Nanjing Kingsrui Biological Technology Co., Ltd. using a solid-phase synthesis method, and the reagent specification is: ≥95.0%, and the amino acid sequence thereof is Cys-Ala-Glu-Lys-Ala; the production manufacturer of dopamine hydrochloride is Aladdin Reagent, 98%; the production manufacturer of tris(hydroxymethyl) aminomethane is Aladdin Reagent, super-pure; the Ti3AlC2 powder is purchased from Shandong Enyan New Material Technology Co., Ltd., and is easy to peel (≥99.5%); the production manufacturer of lithium fluoride is 3A Chemical, 99%; and the reagents or substances not specifically mentioned in the present application are all commercially available products.

[0035] The instruments used in the present application are as follows:

[0036] Transmission electron microscopy (TEM), X-ray diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), infrared spectroscopy and X-ray photoelectron spectroscopy (XPS) are selected to characterize the structure and chemical composition of the MXene-based targeted photothermal nano-reagent MPP.

[0037] The following test section is provided to illustrate the test method; the test section is as follows:

[0038] The model of the TEM is JEM 1400Plus, and the acceleration voltage is 120kV. The MPP colloidal solution with a suitable concentration is dropped on a putong carbon support film, water is absorbed from the other side of the carbon film with filter paper, and then further dried, and observed with a transmission electron microscope.

[0039] The model of the multi-position automatic sampling X-ray diffractometer is X'pert Powder. The scanning angle is 2-90°.

[0040] The model of the atomic force microscope is XE-100.

[0041] The model of the scanning electron microscope is NOVA NANOSEM 430.

[0042] The X-ray photoelectron spectrometer is model ESCALAB XI+.

[0043] The Fourier transform infrared spectrometer is model Nicolet IS50-Nicolet Continuum.

[0044] Example 1

[0045] Preparation of photothermal nanoreagents targeting bacteria

[0046] like Figure 1 The diagram shown is a schematic diagram (a) of the preparation process of the targeted photothermal nanoreagent prepared in Example 1 of the present invention and a schematic diagram (a) of its treatment of bacterial infection in vivo. The specific steps are as follows:

[0047] 1. Single-layer or multi-layer Ti3C2T is prepared by selectively etching the Al layer of the Ti3AlC2MAX phase using a mixed HCl / LiF reagent. x MXene.

[0048] The specific preparation process is as follows: In a polytetrafluoroethylene (PTFE) beaker, dilute a 36%–38% hydrochloric acid solution to a concentration of 9 mol / L with deionized water, resulting in a total volume of 40 mL. Then, add 2 g of LiF and stir magnetically for 1 hour to completely dissolve it into a transparent homogeneous solution. Under ice-water bath protection, slowly add 1 g of Ti3AlC2 powder to the above mixed solution. After the addition is complete, transfer the beaker to a 40°C water bath and continue stirring for 36 hours. After the reaction is complete, centrifuge the resulting mixed solution (3500 rpm, 5 min) and wash several times with deionized water until the pH of the upper suspension is ≥6. Collect the precipitate and redisperse it in approximately 200 mL of deionized water. Sonicate under N2 protection for 2 hours. After sonication, centrifuge the mixture (3500 rpm, 60 min) and collect the upper suspension, which is the few-layer or monolayer Ti3C2T. x MXene colloidal solution. Take 10 mL of this colloidal solution, freeze-dry it into powder, and weigh it to determine the original solution concentration, which is 1.9 mg / mL.

[0049] 2. MXene@PDA (MP) is prepared by in-situ oxidative polymerization of dopamine (DA) on the surface of MXene under alkaline conditions.

[0050] The specific preparation process is as follows: the MXene colloidal solution (1.9 mg / mL) prepared in step 1 is diluted with deionized water to a volume of 80 mL, and the concentration of the MXene diluted solution is 250 μg / mL, then 0.12114 g of tris(hydroxymethyl) aminomethane (Tris, 1 mmol) is added under the action of magnetic stirring to adjust the pH of the solution. Finally, 20 mL of 5 mg / mL dopamine hydrochloride (DA-HCl) aqueous solution is added to the above mixed aqueous solution by automatic sampler at a feeding speed of 0.25 mL / min, and then reacted at room temperature under light protection for 12 h. The final concentration of the system is: C MXene = 200 μg / mL; C Tris = 10 mmol / L (pH = 8.5); C DA-HCl = 1 mg / mL. After the reaction is completed, the obtained reaction mixture is centrifuged (11000 rpm, 10 min) and washed with deionized water for 2-3 times to remove the unreacted DA monomer, and then the collected MXene@PDA precipitate is re-dispersed in a certain volume of deionized water and 10 mL is freeze-dried to determine its concentration, and the concentration is 730 μg / mL.

[0051] 3. MPP is prepared by Michael addition reaction or Schiff base reaction between thiol or amino in the peptide CAEKA and polydopamine under alkaline conditions.

[0052] 45.6 mL of the above MXene@PDA dispersion is taken in a 250 mL round-bottom flask, and 60.57 mg of tris(hydroxymethyl) aminomethane (Tris, 0.5 mmol) is added under magnetic stirring to make it fully dissolved to adjust the pH of the solution; then the mixed solution is bubbled with N2 for 1 h to remove oxygen in the system. Finally, 20 mL of 5 mg / mL peptide CAEKA aqueous solution is slowly added to the above mixed solution by automatic sampler, and the reaction is carried out at room temperature under the protection of N2 for 48 h. After the reaction is completed, the obtained mixture is centrifuged (11000 rpm, 10 min) and washed with deionized water for 2-3 times to remove the unreacted peptide CAEKA and organic base Tris in the system, and the obtained MPP precipitate is re-dispersed in a certain amount of deionized water, and 10 mL is freeze-dried to powder to determine its concentration, and the concentration is 570 μg / mL.

[0053] Example 2

[0054] Preparation of a photo-thermal nano-reagent targeting bacteria

[0055] 1. The Al layer of Ti3AlC2 MAX phase is selectively etched by using a mixed reagent of HCl / LiF to prepare single-layer or few-layer Ti3C2T x MXene.

[0056] The specific preparation process is as follows: a 36% to 38% mass fraction hydrochloric acid solution is diluted with deionized water to a concentration of 9 mol / L and a total volume of 40 mL in a polytetrafluoroethylene beaker; then 2 g of LiF is added, and magnetic stirring is performed for 1 h to completely dissolve it into a transparent homogeneous solution; 2 g of Ti3AlC2 powder is slowly added to the above-mentioned mixed solution under the protection of an ice water bath, and after the sample addition is completed, the beaker is moved to a water bath at 35°C and continues to be stirred for 48 h. After the reaction is completed, the obtained mixed solution is centrifuged (3500 rpm, 5 min) and washed with deionized water several times until the pH of the upper suspension is greater than or equal to 6, the precipitate is collected and re-dispersed in about 200 mL of deionized water, ultrasonic treatment is performed for 2 h under the protection of N2, after the ultrasonic treatment is completed, the mixture is centrifuged (3500 rpm, 60 min) and the upper suspension is collected, which is Ti3C2T x MXene colloidal solution. 10 mL of the colloidal solution was freeze-dried into a powder and weighed to determine the original solution concentration, which was 2 mg / mL.

[0057] 2. MXene@PDA (MP) was prepared by in-situ oxidative polymerization of dopamine (DA) on the surface of MXene under alkaline conditions

[0058] The specific preparation process is as follows: the MXene colloidal solution (2 mg / mL) prepared in step 1 is diluted with deionized water to a volume of 80 mL, and a MXene dilution solution with a concentration of 250 μg / mL is obtained, then 0.12114 g of tris(hydroxymethyl) aminomethane (Tris, 1 mmol) is added to the solution under the action of magnetic stirring to adjust the pH of the solution. Finally, 20 mL of a 10 mg / mL dopamine hydrochloride (DA-HCl) aqueous solution is added to the above-mentioned mixed aqueous solution through an automatic sampler at a feeding speed of 0.25 mL / min, and then the reaction is carried out at room temperature and in the dark for 12 h. The final concentration of the system is: C MXene = 200 μg / mL; C Tris = 10 mmol / L (pH = 8.5); CDA-HCl = 2 mg / mL. After the reaction is completed, the obtained reaction mixture is centrifuged (11000 rpm, 10 min) and washed with deionized water 2 to 3 times to remove unreacted DA monomers, and then the collected MXene@PDA precipitate is re-dispersed in a certain volume of deionized water and 10 mL is freeze-dried to determine the concentration, which is 1 mg / mL.

[0059] 3. MXene@PDA@Peptide (MPP) is prepared by Michael addition reaction or Schiff base reaction between the thiol or amino group in the peptide CAEKA and polydopamine under alkaline conditions.

[0060] Take the above MXene@PDA dispersion 33 mL in a 250 mL round-bottom flask, and dilute with deionized water to 45.6 mL, 60.57 mg of tris(hydroxymethyl)aminomethane (Tris, 0.5 mmol) is added under magnetic stirring, and it is fully dissolved to adjust the pH of the solution; then the mixed solution is bubbled with N2for 1 h to remove oxygen in the system. Finally, 20 mL of 5 mg / mL peptide CAEKA aqueous solution is slowly added to the above mixed solution by automatic sampler, and the reaction is carried out at room temperature for 48 h under the protection of N2. After the reaction is completed, the obtained mixture is centrifuged (11000 rpm, 10 min) and washed with deionized water for 2-3 times to remove unreacted peptide CAEKA and organic base Tris in the system, and the obtained MPP is redispersed in a certain amount of deionized water, and 10 mL of freeze-dried powder is taken to determine the concentration, which is 610 μg / mL.

[0061] Example 3

[0062] Preparation of a photo-thermal nano-reagent targeting bacteria

[0063] 1. Selective etching of the Al layer of Ti3AlC2 MAX phase with an HCl / LiF mixed reagent to prepare single-layer or few-layer Ti3C2T x MXene.

[0064] The specific preparation process is as follows: in a polytetrafluoroethylene beaker, dilute a hydrochloric acid solution with a mass fraction of 36%-38% to a concentration of 9 mol / L with deionized water, and the total volume is 40 mL; then 2 g of LiF is added, and it is fully dissolved into a transparent homogeneous solution under magnetic stirring for 1 h; 2 g of Ti3AlC2 powder is slowly added to the above mixed solution under the protection of an ice-water bath, and after the addition is completed, the beaker is moved to a water bath at 40°C and continues to stir for 24 h. After the reaction is completed, the obtained mixed solution is centrifuged (3500 rpm, 5 min) and washed with deionized water several times until the pH of the upper suspension is ≥6, the precipitate is collected and redispersed in about 200 mL of deionized water, ultrasonic treatment is carried out in an ice-water bath under the protection of N2for 3 h, and after the ultrasonic treatment is completed, the mixture is centrifuged (3500 rpm, 60 min) and the upper suspension is collected, which is a few-layer or single-layer Ti3C2T x MXene colloidal solution. Take 10 mL of the colloidal solution and freeze-dry it into powder to determine the concentration of the original solution, which is 2 mg / mL.

[0065] 2. MXene@PDA (MP) is prepared by in-situ oxidation and polymerization of dopamine (DA) on the surface of MXene under alkaline conditions

[0066] The specific preparation process is as follows: the MXene colloidal solution (2 mg / mL) prepared in step 1 is diluted with deionized water to a volume of 80 mL, and the concentration of the MXene diluted solution is 250 μg / mL, then 0.12114 g of tris(hydroxymethyl) aminomethane (Tris, 1 mmol) is added under the action of magnetic stirring to adjust the pH of the solution. Finally, 20 mL of 10 mg / mL dopamine hydrochloride (DA-HCl) aqueous solution is added to the above mixed aqueous solution by an automatic sampler at a feeding speed of 0.25 mL / min, and then reacted at room temperature under light-proof conditions for 6 h. The final concentration of the system is: C MXene = 500 μg / mL; C Tris = 10 mmol / L (pH = 8.5); CDA-HCl = 2 mg / mL. After the reaction is completed, the obtained reaction mixture is centrifuged (11000 rpm, 10 min) and washed with deionized water for 2-3 times to remove the unreacted DA monomer, and then the collected MXene@PDA precipitate is redispersed in a certain volume of deionized water and 10 mL is freeze-dried to determine the concentration, and the concentration is 500 μg / mL.

[0067] 3, MXene@PDA@Peptide (MPP) is prepared by Michael addition reaction or Schiff base reaction between thiol or amino in peptide CAEKA and polydopamine under alkaline conditions.

[0068] Take 100 mL of the above MXene@PDA dispersion liquid in a 250 mL round-bottom flask, and add 133.25 mg of tris(hydroxymethyl) aminomethane (Tris, 1.1 mmol) under magnetic stirring, and make it fully dissolved to adjust the pH of the solution; then the mixed solution is bubbled with N2 for 1 h to remove oxygen in the system. Finally, 10 mL of 5 mg / mL peptide CAEKA aqueous solution is slowly added to the above mixed solution by an automatic sampler, and the reaction is carried out at room temperature under the protection of N2 for 48 h. After the reaction is completed, the obtained mixture is centrifuged (11000 rpm, 10 min) and washed with deionized water for 2-3 times to remove the unreacted peptide CAEKA and organic base Tris in the system, and the obtained MPP precipitate is redispersed in a certain amount of deionized water, and 10 mL is freeze-dried to powder to determine the concentration, and the concentration is 650 μg / mL.

[0069] II. Data test

[0070] 1. Structure characterization of substances related to example 1

[0071] As Figure 2 shown, Ti3C2T xScanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), infrared spectroscopy and X-ray diffraction data of MXene, MXene@PDA. Figure 2 (a), Figure 2 (b) are single-layer Ti3C2T x Transmission electron microscopy (TEM) images and atomic force microscopy (AFM) images of MXene, MXene@PDA. It is obvious that the contrast of single-layer Ti3C2T x MXene has an ultra-thin atomic layer thickness and small contrast, thus showing a relatively transparent sheet structure. The thickness of MXene was characterized by atomic force microscopy, and the thickness of single-layer Ti3C2T x The thickness of MXene is about 2 nm; when Ti3C2T x After the surface of MXene nanosheet is coated with polydopamine, it can be seen from the transmission electron microscopy image that the contrast of the nanosheet is significantly increased, and the thickness is increased from the original 2 nm to 50 nm to 60 nm. In addition, the Ti3C2T x The surface state of MXene and MXene@PDA was characterized, as shown in Figure 2 (c) and (d), pure Ti3C2T x MXene nanosheet presents a light and thin nanoflower petal, and after surface modification with polydopamine, it can be clearly observed that there are nanoparticles on the surface of the nanosheet, and the sheet layer becomes thick and heavy. From Ti3C2T x The Tyndall effect phenomenon of MXene and MXene@PDA colloidal solution shows that the nanosheet has good dispersibility and nanosize effect in water before and after surface modification, as shown in Figure 2 (e). In addition, Figure 2 (f) is Ti3AlC2 MAX phase and prepared Ti3C2T x X-ray diffraction (XRD) patterns of MXene, polydopamine and MPP. The bulk Ti3AlC2 MAX phase shows very obvious diffraction peaks, and the crystal face parameters corresponding to each diffraction peak are marked in the figure. After etching and ultrasonic exfoliation, the diffraction peak originally located at 39° is greatly weakened and almost disappears, proving that the Al element layer is successfully etched off; in addition, the diffraction peak of Ti3C2(002) crystal face is shifted from 9.5° before etching to 6.3°, which indicates that by selective etching of Ti3AlC2 with HCl / LiF, we successfully obtain Ti3C2T x MXene. When Ti3C2T x After the surface of MXene is coated with a layer of polydopamine, the diffraction peak of Ti3C2(002) crystal face will disappear, because Ti3C2Tx The polydopamine coating on the surface of MXene prevents the self-stacking of monolayer nanosheets. From the Fourier transform infrared spectroscopy spectrum, it can be seen that the absorption peak of 3400cm Figure 2 (g)It can be seen that Ti3C2T x The wave number in MXene, PDA and MXene@PDA is 3400cm -1 Absorption peaks appear near, which is due to the stretching vibration of hydroxyl (-OH) on the surface of MXene nanosheets and amino (N-H) in polydopamine, 1626cm -1 The absorption peak at 1503cm -1 The absorption peak at 624cm -1 The absorption peak at 624cm

[0072] As shown in Figure 1, the X-ray photoelectron spectroscopy (XPS) of the targeted photothermal nanoreagent MXene@PDA@Peptide (MPP) prepared in Example 1 of the present application is shown. Figure 3 It can be observed that, after introducing the peptide CAEKA to the surface of Ti3C2T x MXene, the electron binding energy peak of S element appears in MPP, since each CAEKA contains only one S atom, the peak of S element in the full spectrum is very small, which can be observed by high-resolution X-ray photoelectron spectroscopy. The above shows that we successfully introduced the short peptide chain CAEKA with the function of recognizing bacteria into the surface of Ti3C2T x MXene, and successfully constructed the targeted photothermal nanoreagent MPP.

[0073] 2, Effect test

[0074] Detection method:

[0075] In this experiment, E. coli (ATCC 43894), S. aureus (ATCC 25923) and MRSA (ATCC 29213) were used as models of gram-negative, gram-positive and drug-resistant bacteria, respectively, to study the in vitro photothermal antibacterial activity of nano-reagent MPP. First, the bacteria stored at -80°C were drawn on the solid Luria-Bertani (LB) agar plate by streaking method, and incubated at 37°C in a constant temperature biochemical incubator for 24 hours. A single colony was dispersed in 3 mL of liquid LB medium using a 100 μL pipette tip, and incubated at 200 rpm and 37°C for 12 hours. Then, it was further incubated under the same conditions for 6 hours to reach the logarithmic growth phase. After incubation, 1 mL of bacterial suspension was centrifuged (5000 rpm, 10 min) and washed twice with sterile phosphate buffered saline (PBS) to remove bacterial metabolites. The washed bacterial pellet was then dispersed in 1 mL of sterile PBS for use.

[0076] 20 μL of the above prepared bacterial suspension was taken with a pipette and mixed with different concentrations of Ti3C2T x MXene and MPP colloidal solution were mixed to a final volume of 200 μL, and the sample concentrations were 50 μg / mL, 40 μg / mL, 30 μg / mL, and 20 μg / mL, respectively. Each concentration was set in two groups (light group + dark group). Then the sample and bacterial mixed suspension were incubated in the dark for 30 min, and then the MPP groups of different concentrations were irradiated under near-infrared laser with a wavelength of 808 nm and a light power density of 0.7 W / cm 2 The temperature of the system after 5 min of irradiation was recorded with an infrared thermal imager, and 20 μL was taken for gradient dilution with a dilution factor of 10 3 times. Finally, 100 μL of the diluted bacterial solution was plated and incubated at 37°C in a constant temperature biochemical incubator overnight, and the number of colonies was counted. For the MXene group, the temperature of the system was monitored in real time with an infrared thermal imager to keep it at the same level as the corresponding concentration of the MPP group (the irradiation time did not need to be the same). The remaining procedures were the same as those for the MPP group. 20 μL of the original bacterial solution was diluted 10 4 times, and 100 μL was taken for plating as a blank group.

[0077] As Figure 4 shown in Figure 1, MPP prepared in Example 1 had good in vitro photothermal antibacterial activity against E. coli, S. aureus and MRSA. The killing effect of MPP on E. coli, S. aureus and MRSA was 100%, 99.9% and 99.9%, respectively. Figure 4(a-f) It can be seen that at the same end-state overall temperature level, the bactericidal effect of MPP on E. coli, S. aureus and MRSA is better than that of Ti3C2T x MXene.

[0078] The specific antibacterial result analysis is as follows: when the sample concentration is 40 μg / mL, for E. coli, the antibacterial effect of MPP+NIR group is about 0.27 orders of magnitude better than that of Ti3C2T x MXene+NIR group; for S. aureus, the antibacterial effect of MPP+NIR group is about 0.98 orders of magnitude better than that of Ti3C2T x MXene+NIR group. This is because E. coli, as a representative of gram-negative bacteria, has a thick outer membrane covering the cell wall, which shields the peptidoglycan in the cell wall, resulting in a decrease in the affinity of MPP to bacteria; but because there are membrane proteins in the outer membrane of E. coli, a weak interaction can occur between the peptide chains on the surface of MPP, so the antibacterial effect of MPP is only slightly better than that of Ti3C2T x MXene. In addition, E. coli, as a kind of gram-negative bacteria, contains a substance called lipopolysaccharide (LPS), i.e., endotoxin, in its cell wall, which makes it resistant to heat to some extent. Therefore, the antibacterial effect of MPP is better than that of Ti3C2T x MXene for S. aureus.

[0079] When the sample concentration reaches 50 μg / mL, for E. coli, the antibacterial effect of MPP+NIR group is about 0.93 orders of magnitude better than that of Ti3C2T x MXene+NIR group, and the significant order of magnitude is greatly increased compared with 40 μg / mL, because as the temperature increases, the weak targeting effect of MPP on E. coli will be increasingly obvious; and for S. aureus, the antibacterial effect of MPP+NIR group is about 0.93 orders of magnitude better than that of Ti3C2T xThe antibacterial effect of the MXene + NIR group was about 0.92 orders of magnitude, which was roughly the same as when the sample concentration was 40 pg / mL. This was because, relative to E. coli, S. aureus was inherently more fragile, and even without targeting, the higher system temperature would cause its mortality to be very high. Therefore, the high temperature would make the strong targeting effect of MPP flatten out. Methicillin-resistant Staphylococcus aureus (MRSA) is more survivable than drug-sensitive Staphylococcus aureus (S. aureus) due to its inherent drug resistance, so the targeting effect of MPP is particularly evident (differential effect) at high system temperatures. When the sample concentration was 50 pg / mL, the bactericidal effect of MPP on MRSA was 1.95 orders of magnitude higher than that of Ti3C2T x MXene. In addition, the calculation of the bactericidal rate showed that when the sample concentration was 50 pg / mL, the bactericidal rates of MPP on E. coli, S. aureus and MRSA were 97.85%, 99.87% and 99.51%, respectively. It can be seen that the killing effect of MPP on gram-positive bacteria is better than that on gram-negative bacteria.

[0080] As Figure 5 Targeting effect of MPP prepared in Example 1 on MRSA. As Figure 5 (a) shows that the surface of MRSA without any treatment is smooth and flat, and the surface of MRSA after co-culturing with MPP is shown in (b). Figure 5 (b) It can be seen that the content of Ti element on the surface is negligible; and after co-culturing with MPP, the surface of MRSA cells can be clearly observed to have uneven flaky structures (as indicated by the red arrows in the figure). In order to verify that the flaky structure on the surface of MRSA cells is MPP nanosheet, we further characterized the element distribution on the surface by SEM-EDS. As Figure 5 (b) shows that when there is no co-culturing with MPP, the distribution of Ti element on the surface of MRSA is in the whole area and is not obvious, which can be attributed to the detection error of the instrument. After co-culturing with MPP, as shown in (c), the planar distribution shape of N element and Ti element is highly consistent with the outline of the MPP / MRSA combination in the figure, and the mass fraction of Ti element also increases to 1.73%, which strongly proves that the flaky structure on the surface of MRSA is indeed MPP nanosheet, i.e. MPP has the ability to target MRSA.

[0081] In vitro biocompatibility of MPP prepared in Example 1. Figure 6 In vitro biocompatibility of MPP prepared in Example 1. Figure 6 (a) is Ti3C2T x Hemolysis of MXene and MPP (0.2% (v / v) Triton X-100 aqueous solution as positive control, sterile PBS as negative control group). When Ti3C2Tx When the concentration of MXene colloidal solution reached 100 pg / mL, the supernatant after co-culture with red blood cells showed a distinct red color. This indicates that the Ti3C2T x MXene has shown a more serious hemolysis, and the calculated hemolysis rate is 4.787%. When the concentration of Ti3C2T x MXene increased to 200 pg / mL, the hemolysis rate reached 8.865%, at this time, most of the red blood cells were severely damaged (the liquid red color was significantly deepened). Analysis shows that the Ti3C2T x MXene has a sharp edge. As a new type of graphene-like two-dimensional nanomaterial, Ti3C2Tx MXene has an atomic level thickness, but its lateral size can reach 300-500 nm, so the large aspect ratio makes it have a sharp edge. When it contacts with red blood cells, Ti3C2T x MXene acts like a sharp nanometer knife to cut the membrane of red blood cells, causing hemoglobin to leak. Just as it can destroy the integrity of bacterial cells through its sharp edge, thereby achieving bactericidal effect. In this study, Ti3C2T x MXene was surface modified with polydopamine and peptide CAEKA. Compared with simple Ti3C2T x MXene, the blood compatibility of the obtained MPP is significantly improved. Within the entire concentration range of MPP set, the color change of the supernatant after co-culture with red blood cells is not obvious. After calculating the hemolysis rate, it is found that when the concentration is 200 pg / mL, the hemolysis rate of MPP is less than one-tenth of that of MXene, and when the concentration of MPP is 100 pg / mL and below, its hemolysis rate is very low, all within 0.025%, showing good blood compatibility. In addition, we used mouse fibroblasts (L929) as the target, and evaluated the cytotoxicity of materials by co-culturing with Ti3C2T x MXene and MPP. As shown in Fig. Figure 6 (b), when the sample concentration is in the range of 20-100 pg / mL, the cell survival rate of the MPP group is always greater than that of the Ti3C2T x MXene group. This shows that after surface modification, the cytotoxicity of MPP is greatly reduced compared with the original Ti3C2T x MXene. Finally, we showed through the live and dead cell staining experiment that after co-culture with mouse fibroblasts, the cell activity in the MPP group is higher (the green fluorescence is stronger); while for Ti3C2T x MXene, when the concentration is greater than or equal to 80 pg / mL, the activity of the cells is greatly reduced (the red fluorescence gradually becomes the dominant color).

[0082] Based on our analysis, we have summarized the reasons for the good biocompatibility of MPP as follows: First, polydopamine was used to modify Ti3C2T... x After surface modification, the sharp edges of MXene nanosheets are blunted by the coating with polydopamine. Therefore, when MPP comes into direct contact with normal cells, its mechanical destructive effect is greatly reduced. Secondly, polydopamine, as a biomimetic material, has good biocompatibility; therefore, after surface modification with polydopamine, Ti3C2T… x The toxic effects of MXene on cells were significantly reduced. Finally, the peptide CAEKA we selected contains cysteine, alanine, glutamic acid, and lysine, all four of which are major amino acids that make up proteins in organisms. Therefore, the introduction of this peptide further improved the performance of Ti3C2T. x Biocompatibility of MXene. In vitro antibacterial data show that when the concentration of MPP is 50 μg / mL, at 808 nm and 0.7 W / cm², [the following data is presented:] MXene biocompatibility. 2 Irradiation with near-infrared light for 5 min resulted in bactericidal rates of 97.85%, 99.87%, and 99.51% against E. coli, S. aureus, and MRSA, respectively, demonstrating excellent bactericidal effects. This indicates that the targeted photothermal nanoreagent MPP we constructed exhibits good biocompatibility within a concentration range with significant bactericidal effects, combining effective bactericidal properties with good biocompatibility.

[0083] like Figure 7 The therapeutic effect of the MPP prepared in Example 1 on subcutaneous abscesses caused by MRSA infection. Figure 7 (b)–7(e) MPP effectively eliminated abscesses and significantly reduced the number of bacteria at the site of infection. H&E staining images of skin tissue showed no significant differences in the number and morphology of hair follicles among the different groups after treatment with different samples. Furthermore, in the PBS group, vancomycin group (Van), and Ti3C2T group… x In the MXene+NIR group, infiltration of inflammatory cells (such as lymphocytes and eosinophils) was observed in both the epidermis and dermis (as indicated by the small red arrows in the figure); however, no inflammatory cells were observed in the MPP+NIR group. In Masson's trichrome stained sections of skin tissue, the PBS group and the Ti3C2T group showed... xNo obvious fibrous tissue proliferation was observed in the MXene+NIR group; while in the Van group, fibrous tissue proliferation was observed in the subcutaneous fat layer; in the MPP+NIR group, fibrous tissue proliferation was observed in the subcutaneous fat layer. The above pathological results show that the target photothermal nano reagent MPP constructed by us can effectively treat subcutaneous abscess caused by methicillin-resistant Staphylococcus aureus infection, and the healing of the infection site is better after MPP+NIR treatment.

[0084] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. In addition, the present application can be implemented in other ways, although the present application has been described in detail with reference to the above examples. Those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents, but these modifications or replacements are within the protection scope of the present application.

Claims

1. A method for preparing a photothermal nanoagent targeting bacteria, characterized in that, Comprising the following steps: S1, Ti3AlC2 clay as raw material, with etchant for selective etching of Al element layer, inert gas protection under ultrasonic, Ti3C2T x MXene nanosheet; S2, under alkaline conditions, Ti3C2T x Surface modification of MXene nanosheets to form a polydopamine coating to obtain MXene@PDA; S3, under alkaline conditions, short-chain peptides capable of identifying bacteria are grafted to the surface of MXene@PDA obtained in S2 under inert gas protection to obtain MXene@PDA@Peptide; the amino acid sequence of the short-chain peptide is cysteine-alanine-glutamic acid-lysine-alanine.

2. The production method according to claim 1, characterized by, The etchant is HCl / LiF, the concentration of HCl is 8-10 mol / L, and the concentration of LiF is 0.04-0.06 g / mL.

3. The preparation method according to claim 2, characterized in that, The etching temperature is 40±1℃, and the etching time is 36±0.5h.

4. The production method according to claim 1 or 2, characterized by, The ratio of Ti3AlC2 clay to etchant in S1 is (1-2) g:(30-40) ml.

5. The production method according to claim 1 or 2, characterized by, S2 on Ti3C2T x The substance for surface modification of the MXene nanoplatelets is dopamine hydrochloride, with a concentration ranging from 1 to 2 mg / mL.

6. The production method according to claim 1 or 2, characterized by, The reaction time of S2 is 12±0.1h; it is carried out in a dark environment.

7. The preparation method according to claim 6, characterized in that, The pH of the alkaline aqueous solution in S2-S3 is 8.3-9.0, and the pH value is adjusted by tris(hydroxymethyl) aminomethane or sodium hydroxide.

8. The method of claim 1, wherein, The concentration of the short-chain peptide is 0.5-2 mg / mL.

9. The production method according to claim 1 or 8, characterized by, The short-chain peptide is grafted to the surface of MXene@PDA obtained in S2 by Michael addition reaction or Schiff base reaction between thiol or amino and polydopamine, and the mass ratio of MXene@PDA obtained in S2 to the short-chain peptide is 1:(1-3).

10. The method of claim 1, wherein, The bacteria in S3 are selected from gram-negative bacteria and gram-positive bacteria.

11. The preparation method according to claim 10, characterized in that, The bacteria are selected from Escherichia coli and Staphylococcus aureus.

12. The method of claim 10, wherein, The bacteria are selected from methicillin-resistant Staphylococcus aureus.

13. Use of the photothermal nano-reagent prepared according to any one of claims 1-12 in the preparation of an antibacterial infection drug.

14. Use according to claim 13, characterized in that, The bacteria are methicillin-resistant Staphylococcus aureus.

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