Composite bacteriostatic nanomaterial, and preparation method and application thereof

Abstract

The application discloses a composite bacteriostatic nanomaterial and a preparation method and application thereof, and belongs to the technical field of medical materials. The composite bacteriostatic nanomaterial takes molybdenum disulfide nanosheets as a drug carrier, and loads antibacterial peptides and azobis diisopropyl imidazoline hydrochloride. The composite bacteriostatic nanomaterial can synergistically exert the functions of each component, can make AIPH crack to generate alkyl radicals by using the photo-thermal effect of MoS2, can more effectively kill bacteria by using the dual treatment effect of photo-thermal and chemical, and has excellent bacteriostatic effect. Meanwhile, the composite bacteriostatic nanomaterial can reduce the hemolysis rate of the antibacterial peptides, has few side effects, and is high in safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical materials, and particularly relates to a composite bacteriostatic nanomaterial and a preparation method and application thereof. BACKGROUND

[0002] Bacterial infection seriously threatens human life and health, and traditional antibiotic treatment is one of the main means for clinically treating bacterial infection. However, the use of traditional antibiotics in large quantities can easily make bacteria resistant, and a new treatment method needs to be developed to reduce the use of antibiotics.

[0003] Antibacterial peptides (AMPs) have broad-spectrum antibacterial activity, small size, and easy metabolism, making them an attractive potential antibiotic replacement therapy. Amphiphilic cationic antibacterial peptides act on the bacterial cell membrane through charge, destroy its stability, and achieve antibacterial effect, and have the advantage of not being prone to drug resistance. However, free antibacterial peptides have poor stability and also have certain hemolytic activity, which can cause red blood cells to rupture, so that the clinical application of antibacterial peptides has been greatly limited. SUMMARY

[0004] 1. Invention purposes

[0005] The purpose of the present application is to provide a composite bacteriostatic nanomaterial and a preparation method and application thereof, which uses molybdenum disulfide nanosheets (MoS2) as a drug carrier, and loads antibacterial peptides (AMPs) and azobis diisopropylimidazoline hydrochloride (AIPH). The composite bacteriostatic nanomaterial can synergistically exert the functions of each component, utilize dual photothermal and chemical treatment, and more effectively kill bacteria, thereby having excellent antibacterial effect. At the same time, the hemolysis rate of the antibacterial peptides can be reduced, and the composite bacteriostatic nanomaterial has less side effects and high safety.

[0006] 2. Technical solutions

[0007] In order to achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0008] The present application provides a composite bacteriostatic nanomaterial, which uses molybdenum disulfide nanosheets as a drug carrier, and loads antibacterial peptides and azobis diisopropylimidazoline hydrochloride, wherein:

[0009] Molybdenum disulfide nanosheets (MoS2) have good physical, chemical, and optical properties, are excellent nanomaterial carriers, and also have high photothermal performance, and are a photothermal bacteriostatic material;

[0010] Azobis diisopropylimidazoline hydrochloride (AIPH) is a non-oxygen-dependent alkyl radical initiator, and under anoxic conditions, heat initiates the cleavage of AIPH to generate alkyl radicals;

[0011] The antibacterial peptide (AMP) is loaded by molybdenum disulfide nanosheets to improve the stability, reduce the hemolysis rate and improve the safety.

[0012] The composite antibacterial nanomaterial excites the molybdenum disulfide nanosheet to produce heat under the light irradiation such as near-infrared light, causing the azo diisopropyl imidazoline hydrochloride to crack to produce alkyl radicals, so as to achieve the combined antibacterial effect.

[0013] Further, the amino acid sequence of the antibacterial peptide in the composite antibacterial nanomaterial is G (IIKK) 3-I-NH2.

[0014] Further, the composition (weight percentage) of each component in the composite antibacterial nanomaterial is as follows: MoS2, 50% to 55%; AMP, 20% to 25%; AIPH, 20% to 30%.

[0015] Further, the composition (weight percentage) of each component in the composite antibacterial nanomaterial is as follows: MoS2, 55%; AMP, 22%; AIPH, 23%.

[0016] Further, the composition (weight percentage) of each component in the composite antibacterial nanomaterial is as follows: MoS2, 52%; AMP, 23%; AIPH, 25%.

[0017] The application also provides a preparation method of the composite antibacterial nanomaterial, which comprises the following steps:

[0018] S1, preparation of MoS2-AMP: MoS2 nanosheets and AMP are mixed in an aqueous solution, stirred and centrifuged to obtain a precipitate, the precipitate is dissolved in water and centrifuged again, and the precipitate is collected to obtain MoS2-AMP;

[0019] S2, preparation of MoS2-AMP-AIPH: AIPH is added to the solution of MoS2-AMP in S1, and stirred uniformly to prepare MoS2-AMP-AIPH antibacterial material.

[0020] Further, in S1, the mass ratio of MoS2 to AMP is 2:1.

[0021] Further, in S1, the stirring speed is 250 to 300 rpm, and the stirring reaction time is 6 to 8 h.

[0022] Further, in S1, the stirring is magnetic stirring.

[0023] Further, in S1, the centrifugation speed is 8000 to 12000 rpm, and the centrifugation time is 10 to 15 min.

[0024] Furthermore, in the above S1, the preparation of MoS2 nanosheets includes: adding MoS2 solid powder to an isopropanol solution to obtain a mixed dispersion system; ultrasonically treating the dispersion system in a water bath, centrifuging the obtained solution, collecting the supernatant and performing rotary evaporation to obtain exfoliated MoS2 nanosheets.

[0025] Furthermore, the concentration of molybdenum disulfide in the above dispersion system is 5-10 mg / mL. With this dispersion system, molybdenum disulfide can be stripped more fully.

[0026] Furthermore, the ultrasonic treatment time is 1 to 1.5 hours, and the temperature is not higher than 35°C.

[0027] Furthermore, the solution is centrifuged at a speed of 8000 to 12000 rpm, and the centrifugation time is 10 to 15 minutes.

[0028] Furthermore, the rotation speed of the rotary evaporation is 60-70 rpm, and the evaporation temperature is 55° C.-65° C.

[0029] Furthermore, in the above S2, the concentration of the AIPH solution is 5-10 mg / mL, and the concentration of the MoS2-AMP is 5-10 mg / mL.

[0030] Furthermore, in the above S2, the stirring reaction time is 8 to 12 hours.

[0031] The present application also provides the use of the above-mentioned composite antibacterial nanomaterial and / or the preparation method of the above-mentioned composite antibacterial nanomaterial in antibacterial applications.

[0032] Furthermore, in the above-mentioned antibacterial application, the inhibited bacteria include Staphylococcus aureus and / or Escherichia coli.

[0033] The present application also provides the use of the above-mentioned composite antibacterial nanomaterial and / or the preparation method of the above-mentioned composite antibacterial nanomaterial in the preparation of drugs for treating bacterial infections.

[0034] The present application also provides a pharmaceutical composition comprising the above-mentioned composite antibacterial nanomaterial.

[0035] 3. Beneficial effects

[0036] Compared with the prior art, the present application has the following advantages:

[0037] (1) The application provides a composite bacteriostatic nanomaterial and a preparation method and application thereof. The composite bacteriostatic nanomaterial takes molybdenum disulfide nanosheets as a drug carrier, and loads antibacterial peptides and azobis diisopropyl imidazoline hydrochloride. The AMP and AIPH have strong bactericidal effects on bacteria, and the MoS2 has good photo-thermal conversion efficiency. The composite bacteriostatic nanomaterial can synergistically exert the functions of the components, can kill bacteria more effectively by using photo-thermal and chemical dual treatment effects, has excellent bacteriostatic effects, can reduce the hemolytic effect of the antibacterial peptides on blood cells, and has the advantages of safety and economy, strong targeting effect, strong bacteriostatic capacity, high stability, and few side effects.

[0038] (2) The application provides a composite bacteriostatic nanomaterial and a preparation method and application thereof. The composite bacteriostatic nanomaterial obtains thin-layer MoS2 nanosheets by using an ultrasonic liquid-phase exfoliation method, obtains AMP by using a solid-phase synthesis method, then reacts the AMP with the MoS2 to obtain MoS2-AMP, and then adds an AIPH solution to obtain MoS2-AMP-AIPH, so that the preparation method is simple and convenient. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 FIG. 1 is a synthesis process schematic diagram of the composite bacteriostatic nanomaterial MoS2-AMP-AIPH of the application.

[0040] Figure 2 FIG. 2 is a scanning electron microscope image of the MoS2 nanosheets prepared in the application.

[0041] Figure 3 FIG. 3 is ultraviolet and infrared spectra of various materials prepared in the application, wherein I is an ultraviolet spectrum, and II is an infrared spectrum.

[0042] Figure 4 FIG. 4 is a standard curve of AMP and AIPH, wherein I is AMP, and II is AIPH.

[0043] Figure 5 FIG. 5 is a photo-thermal curve of the composite bacteriostatic nanomaterial MoS2-AMP-AIPH prepared in the application, wherein I is temperature change under 808 nm infrared 0.5 W, 1 W, 1.5 W and 2 W power irradiation, and II is temperature change of different concentrations of MoS2-AMP-AIPH under 808 nm infrared 2 W power irradiation.

[0044] Figure 6 FIG. 6 is an in-vitro antibacterial test effect diagram of the composite bacteriostatic nanomaterial MoS2-AMP-AIPH prepared in the application, wherein I is a blank group, II is a MoS2 group, and III is a drug material group.

[0045] Figure 7The figure is the bacteriostatic rate of the composite bacteriostatic nanomaterial MoS2-AMP-AIPH prepared in the application under fluorescence microscope observation.

[0046] Figure 8 The figure is the hemolysis result of the composite bacteriostatic nanomaterial MoS2-AMP-AIPH prepared in the application and AMP. DETAILED DESCRIPTION

[0047] The application will be further described below in combination with specific examples.

[0048] It should be noted that the terms such as "upper", "lower", "left", "right", "intermediate" and the like cited in the present specification are only for the convenience of clear description, and are not intended to limit the scope of implementation, and the change or adjustment of the relative relationship is also regarded as the scope of implementation of the present application without substantial change of the technical content.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs; the term "and / or" used herein includes any and all combinations of one or more related listed items.

[0050] Unless otherwise specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0051] As used herein, the term "about" is used to provide flexibility to a given term, measurement, or value associated with it. The degree of flexibility of a specific variable can be readily determined by one skilled in the art.

[0052] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations thereof.

[0053] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted -flexibly to avoid construing away readily understandable content unnecessarily to preclude meaning active from this description. For example, a numerical range expressed as "from about 1 to about 4.5" should be interpreted as including not only the explicitly-stated limits of 1 and 4.5, but also implicitly to include individual numbers within the range, for example, and sub-ranges between the explicitly-stated limits of 1 and 4.5, for example, 1 to 4.5, 2.3 to 4.1, etc. The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5." Such a range is to be interpreted to include all individual values and sub-ranges, for example, up to and including the value stated.

[0054] Example 1

[0055] The present example provides a preparation method of composite bacteriostatic nanomaterial MoS2-AMP-AIPH and the composite bacteriostatic nanomaterial MoS2-AMP-AIPH prepared thereby. The synthesis process is shown in Figure 1 and specifically includes the following steps:

[0056] (1) Preparation of MoS2 nanosheets

[0057] Take 1.0 g of unpeeled MoS2 black powder into a 250 mL conical flask, and add 100 mL of isopropyl alcohol into the conical flask. Ultrasonically treat the above dispersion system in a water bath for 1 h, and the liquid temperature of ultrasonic treatment should not be higher than 35°C. Centrifuge the obtained solution at 10000 rpm for 10 min, and then directly collect the supernatant. Treat the supernatant by a rotary evaporator at 60°C and 65 rpm, and finally obtain a thin film to obtain MoS2 nanosheets.

[0058] (2) Preparation of MoS2-AMP

[0059] After weighing, redispersed the MoS2 nanosheets in deionized water to obtain a water solution of peeled MoS2 nanosheets.

[0060] Add 10 mL of 0.005 g / mL MoS2 nanosheet water solution into a container with a magnetic rotor, and then weigh 0.025 g of AMP (G(IIKK)3-I-NH2) and add into the container. Place it on a magnetic stirrer at 250 rpm, and magnetically stir the reaction for 8 h. After the stirring is completed, centrifuge at 12000 rpm for 15 min, collect the precipitate, dissolve in deionized water, and then centrifuge at 12000 rpm for 15 min to collect the precipitate to obtain MoS2-AMP. Detect the absorbance of the supernatant at 220 nm to calculate the loading rate of AMP.

[0061] (3) Preparation of MoS2-AMP-AIPH

[0062] Into a vessel equipped with a magnetic rotor, 5 mL of 0.01 g / mL MoS2-AMP aqueous solution was added. Then, 0.025 g of AIPH was added. The reaction was carried out on a magnetic stirrer at 300 rpm for 8 h. After stirring, the precipitate was collected by centrifugation at 12000 rpm for 15 min, and then dissolved in deionized water and centrifuged at 12000 rpm for 15 min to collect the precipitate, to obtain MoS2-AMP-AIPH. The absorbance of the supernatant at 374 nm was detected for calculating the loading rate of AIPH.

[0063] Result analysis:

[0064] The scanning electron microscope image of the MoS2 nanosheet prepared in this example is shown in Figure 2 The thickness of the MoS2 nanosheet is generally less than 100 nm, and the distribution is good, the thickness is uniform, and the heat conduction efficiency can be better.

[0065] The ultraviolet and infrared spectra of the MoS2-AMP-AIPH prepared in this example are shown in Figure 3 From the peak position in Figure 3 , it can be seen that the material prepared in this example has been successfully modified.

[0066] The standard curve of AMP and AIPH is shown in Figure 4 (I is AMP, and II is AIPH), and the composition (weight percentage) of each component of the MoS2-AMP-AIPH prepared in this example is calculated as follows: MoS2, 55%; AMP, 22%; and AIPH, 23%.

[0067] Example 2

[0068] This example provides a preparation method of the composite bacteriostatic nanomaterial MoS2-AMP-AIPH and the composite bacteriostatic nanomaterial MoS2-AMP-AIPH prepared thereby. Specifically, the method comprises the following steps:

[0069] (1) Preparation of MoS2 nanosheet

[0070] 0.5 g of unexfoliated MoS2 black powder was taken into a 250 mL conical flask, and 100 mL of isopropyl alcohol was added into the conical flask. The above dispersion system was ultrasonically treated in a water bath for 1.5 h, and the liquid temperature of ultrasonic treatment should not be higher than 35℃. The obtained solution was centrifuged at 12000 rpm for 15 min, and then the supernatant was directly collected. The supernatant was treated by a rotary evaporator at 55℃ and 70 rpm, and finally a thin film was obtained, to obtain MoS2 nanosheet.

[0071] (2) Preparation of MoS2-AMP

[0072] After weighing, the MoS2 nanosheets were re-dispersed in deionized water to obtain the exfoliated MoS2 nanosheets aqueous solution.

[0073] Into a container with a magnetic rotor, 10 mL of 0.005 g / mL MoS2 nanosheet aqueous solution was added, and then 0.025 g of AMP(G(IIKK)3-I-NH2) was weighed and added into the container. It was placed on a magnetic stirrer at 300 rpm for magnetic stirring reaction for 6 h. After stirring, it was centrifuged at 8000 rpm for 10 min, and the precipitate was collected and dissolved in deionized water, and then centrifuged at 8000 rpm for 10 min to collect the precipitate to obtain MoS2-AMP. The absorbance of the supernatant at 220 nm was detected for calculating the loading rate of AMP.

[0074] (3) Preparation of MoS2-AMP-AIPH

[0075] Into a container with a magnetic rotor, 5 mL of 0.005 g / mL MoS2-AMP aqueous solution was added. Then, 0.05 g of AIPH was added. It was placed on a magnetic stirrer at 300 rpm for magnetic stirring reaction for 12 h. After stirring, it was centrifuged at 8000 rpm for 10 min, and the precipitate was collected and dissolved in deionized water, and then centrifuged at 8000 rpm for 10 min to collect the precipitate to obtain MoS2-AMP-AIPH. The absorbance of the supernatant at 374 nm was detected for calculating the loading rate of AIPH.

[0076] Result analysis:

[0077] The composition (weight percentage) of each component of the MoS2-AMP-AIPH prepared in this example is as follows: MoS2, 52%; AMP, 23%; AIPH, 25%.

[0078] Example 3

[0079] This example provides photothermal analysis of the MoS2-AMP-AIPH prepared in this application.

[0080] The temperature change of MoS2-AMP-AIPH was measured by measuring the temperature change of MoS2-AMP-AIPH under different concentrations (50, 100, 150, 200 μg / mL, 2.0 W / cm 2 ), different power densities (0.5, 1.0, 1.5, 2.0 W / cm 2 , 150 μg / mL) of 808 nm near-infrared irradiation for 15 min, and the results are shown in Figure 5 As the irradiation time increases, the solution temperature gradually rises, and reaches the upper limit in about ten minutes, and the temperature rise is proportional to the concentration and the irradiation power, indicating that the MoS2-AMP-AIPH prepared in this application has good photo-thermal conversion efficiency.

[0081] Example 4

[0082] This example provides the in-vitro bacteriostatic test of MoS2-AMP-AIPH prepared in this application.

[0083] Using drug-resistant Staphylococcus aureus and Escherichia coli as the target bacteria, the CFU method was used. When the Staphylococcus aureus and Escherichia coli reached the logarithmic phase, they were diluted, and 10 5 0.5 mL of bacterial solution with a concentration of 150 μg / mL of each material was mixed and subjected to light irradiation test under the conditions of 808 nm near-infrared 1.5 W, 5 min. Three repeated groups were made.

[0084] The in-vitro bacteriostatic test results are shown in Figure 6 The Control is the blank control group; the AMP is the antibacterial peptide control group; the MoS2+NIR is the MoS2 material; the MA+NIR is the MoS2-AMP material; and the MAA+NIR is the MoS2-AMP-AIPH material. As can be seen from the figure, compared with MoS2 and MoS2-AMP, MoS2-AMP-AIPH has a significant bacteriostatic effect under light irradiation. At the same time, compared with AMP, the MoS2-AMP-AIPH prepared in this application also has a better effect, and MoS2-AMP-AIPH significantly reduces the amount of antibacterial peptide while achieving a bacteriostatic effect, and also reduces the hemolysis rate caused by the antibacterial peptide.

[0085] Example 5

[0086] This example provides the cell staining fluorescence test of Staphylococcus aureus treated by MoS2-AMP-AIPH prepared in this application.

[0087] Using Staphylococcus aureus as the test strain, the logarithmic phase bacteria were incubated with the drug solution for 1 h, and then cultured at 37°C, 200 r / min for 12 h. Cells treated with other materials at the same concentration were used as the control group, and cells without any treatment were used as the blank group (1 is the blank control group, 2 is the MoS2 material group, 3 is the AMP group, 4 is the MoS2-AMP drug group, and 5 is the MoS2-AMP-AIPH drug group).

[0088] After the culture was completed, the cells were treated with an 808 nm laser emitter at 1.5 W / cm 2, 5 min, and the bacterial cells were collected and washed twice with PBS (0.1 M) to remove the culture medium. The bacteria were stained with a LIVE / DEAD bacterial kit (SYTO9 and propidium iodide (PI), Life Technologies) in the dark for 30 min, and after staining, the cells were washed twice with PBS (0.1 M) to remove the excess staining solution. Finally, the observation was performed on an upright fluorescence microscope, and the results are shown in Figure 7 , the MoS2 group can only inhibit a small part of the bacterial growth under NIR irradiation, but the AMP drug group and the final MoS2-AMP-AIPH group can inhibit most of the bacterial growth under NIR.

[0089] Example 6

[0090] This example provides a cell hemolysis test of MoS2-AMP-AIPH prepared in this application.

[0091] Fresh rat red blood cells (RBC) were washed with PBS buffer for 3 times, and the cell particles were resuspended in PBS buffer to reach about 4% (v / v) suspension. Then, the RBC suspension (1.0 mL) was mixed with 1.0 mL of different concentrations of samples (MoS2-AMP-AIPH and AMP), and the measurement was carried out in triplicate. The mixture was incubated at 37°C for different times (1, 12 or 24 h). All samples were centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected, and all the supernatant of the test samples was added to a 96-well plate. The absorbance of the samples at 570 nm was measured on a microplate reader. The RBC suspension incubated with PBS and 0.1% (v / v) Triton X-100 was used as negative control (NC) and positive control (PC), respectively. The hemolysis percentage was calculated by the following formula: Hemolysis percentage = (absorbance of sample - absorbance of NC) / (absorbance of PC - absorbance of NC) x 100.

[0092] The results are shown in Figure 8 As shown, the hemolysis rate of MoS2-AMP-AIPH at 125 μg / mL was 1.7% after 24 h, and the hemolysis rate of MoS2-AMP-AIPH at 250 μg / mL was 3.2%, and under the same concentration conditions, the hemolysis rates of AMP were 17% and 31%, respectively, far exceeding the safety value of 5%, and the hemolysis percentage of MoS2-AMP-AIPH prepared in this application was low, and the safety was good.

Claims

1. A composite antibacterial nanomaterial, characterized in that: The composite antibacterial nanomaterial uses molybdenum disulfide nanosheets MoS2 as a drug carrier, loaded with antimicrobial peptide AMP and azobisisopropylimidazoline hydrochloride AIPH; the amino acid sequence of the antimicrobial peptide AMP is: G(IIKK)3-I-NH2; The preparation method of the composite antibacterial nanomaterial comprises the following steps: S1, preparation of MoS2-AMP: MoS2 nanosheets and AMP were mixed in aqueous solution, stirred and centrifuged to obtain a precipitate, the precipitate was dissolved in water and centrifuged again, and the precipitate was collected to obtain MoS2-AMP; S2, preparation of MoS2-AMP-AIPH: add AIPH to the MoS2-AMP solution in S1, stir evenly, and prepare MoS2-AMP-AIPH antibacterial material.

2. A composite antibacterial nanomaterial according to claim 1, characterized in that: The weight percentages of the components in the composite antibacterial nanomaterial are: MoS2, 50% to 55%; AMP, 20% to 25%; and AIPH, 20% to 30%.

3. A composite antibacterial nanomaterial according to claim 2, characterized in that: The mass ratio of MoS2 to AMP in the S1 is 2:

1.

4. The composite antibacterial nanomaterial according to claim 3, characterized in that: In the S1, the stirring speed is 250-300 rpm, and the stirring reaction time is 6-8 h; the centrifugal speed is 8000-12000 rpm, and the centrifugation time is 10-15 min.

5. A composite antibacterial nanomaterial according to claim 3 or 4, characterized in that: The preparation of the MoS2 nanosheets includes: adding MoS2 solid powder to an isopropyl alcohol solution to obtain a mixed dispersion system; ultrasonically treating the dispersion system in a water bath, centrifuging the obtained solution, collecting the supernatant and performing rotary evaporation to obtain exfoliated MoS2 nanosheets.

6. The composite antibacterial nanomaterial according to claim 5, characterized in that: In the preparation of the MoS2 nanosheets, the concentration of molybdenum disulfide in the dispersed system is 5~10 mg / mL; and / or the ultrasonic treatment time is 1~1.5 h, and the temperature is not higher than 35°C; and / or the centrifugal speed is 8000~12000 rpm, and the centrifugal time is 10~15 min; and / or the rotary evaporation speed is 60~70 rpm, and the evaporation temperature is 55°C~65°C.

7. Use of the composite antibacterial nanomaterial according to any one of claims 1 to 6 in the preparation of a drug for treating bacterial infection.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a composite antibacterial nanomaterial according to any one of claims 1 to 6.

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