A synergistic antibacterial nano-platform and a method for constructing the same

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a synergistic antibacterial nano platform and a construction method thereof. The nano platform comprises Pec@PLL-MoS2 nanosheets composed of pectinase Pec, epsilon-polylysine PLL and MoS2 nanosheets. The application has the beneficial effects that the nano platform can be stably dispersed in an aqueous solution and has low cytotoxicity, can realize programmed eradication of biofilm infection through photo-thermal mediated, fast controllable EPS enzymolysis, excellent biofilm permeability and broad-spectrum and high-efficiency photo-thermal sterilization performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a synergistic antibacterial nano-platform and a method for constructing the same. BACKGROUND

[0002] Although antibiotics are currently the first choice for the treatment of bacterial infections in the clinic, the abuse of antibiotics has led to the development of drug resistance in many bacteria. More seriously, mature biofilms inhibit the penetration of antibiotics, so the drug resistance of biofilms is 10-1000 times higher than that of free bacteria.

[0003] Biofilm infection occurs when a large number of bacteria adhere to the surface of a wound and are wrapped by extracellular polymers. Extracellular polymers composed of extracellular polysaccharides (EPS), proteins and extracellular DNA (eDNA) play a crucial role in biofilms. They promote biofilm adhesion, act as a barrier against microbial agents (including antibiotics), and use modification enzymes to degrade antibiotics. Notably, EPS is the main component of extracellular polymer substances. In addition to increasing the adhesion and aggregation of biofilms, EPS is the most important component of the protective barrier of biofilms against antibacterial agents. Photodynamic therapy, photothermal therapy and biofilm dispersinase have been widely used to remove polymer substances by the effects of oxidation, thermal denaturation and enzymolysis, respectively. In the above strategies, the use of glycoside hydrolases has recently attracted considerable attention. This is because glycoside hydrolases can degrade EPS, thereby dispersing biofilms and promoting the penetration of antibacterial agents. For example, amylase degrades about 60% of EPS in methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa biofilms by cleaving alpha-1,4-glycosidic bonds. Pec is a type of glycoside hydrolase with polygalacturonase, pectinase, cellulase and hemimethylcellulase activity, which can effectively degrade more than 90% of EPS in S. aureus and E. coli biofilms. These studies show that the use of glycoside hydrolases to degrade EPS is an effective strategy to inhibit and remove biofilms. However, it is known that temperature significantly affects enzymatic reactions, with maximum enzyme activity obtained at a specific optimal temperature. Glycoside hydrolases lack antibacterial properties, so they cannot eradicate biofilms. In order to eliminate biofilms by enzymolysis, it is necessary to combine a non-antibiotic therapy with strong antibacterial activity and provide an optimal temperature for glycoside hydrolases.

[0004] Photothermal therapy (PTT) is a new type of non-antibiotic antibacterial method, which uses photothermal materials that can convert light into heat to achieve sterilization by inducing local hyperthermia through the complete irradiation of an external laser source (usually near-infrared laser). PTT provides a series of benefits, including non-invasive treatment, spatiotemporal control, broad-spectrum antibacterial activity, and minimal bacterial resistance compared to antibiotics. Commonly used photothermal materials include gold nanoparticles, graphene oxide, indocyanine green, and nano-MoS2. MoS2 nanosheets (NSs) are widely used to treat various bacterial infections due to their high photothermal conversion efficiency, good biosafety, and ease of surface modification. In addition, MoS2 NSs not only have excellent antibacterial effect, but also provide optimal temperature, thereby enhancing the enzymatic hydrolysis of EPS and the permeability of antibacterial drugs.

[0005] Currently, there is no report on the construction of Pec cooperated with MoS2 nanosheets and the corresponding performance research in the domestic and foreign literature, so a synergistic antibacterial nano platform is proposed to be applied to the treatment of wound infection. SUMMARY

[0006] In view of the deficiencies in the prior art, the purpose of the present application is to provide a synergistic antibacterial nano platform and a construction method thereof to solve the above problems.

[0007] The technical scheme of the present application is as follows: a synergistic antibacterial nano platform, the nano platform comprising Pec@PLL-MoS2 nanosheets composed of pectinase Pec, epsilon-polylysine PLL and MoS2 nanosheets.

[0008] Further, a construction method of a synergistic antibacterial nano platform, comprising the following steps:

[0009] Step one, MoS2 nanosheets MoS2 NSs are obtained by ultrasonic liquid phase exfoliation method in N-methyl pyrrolidone NMP solution to exfoliate large-diameter MoS2;

[0010] Step two, centrifugal removal of NMP solution and washing with water twice;

[0011] Step three, removing the hydrogen chloride molecules in epsilon-polylysine hydrochloride PLL·HCl by sodium hydroxide to obtain epsilon-polylysine PLL;

[0012] Step four, mixing PLL with MoS2 nanosheets and stirring at room temperature, so that PLL is adsorbed onto MoS2 NSs through electrostatic interaction;

[0013] Step five, centrifugal removal of unreacted PLL in step four and washing with water twice to obtain PLL-MoS2.

[0014] Step six, PLL-MoS2 obtained in step five is added to Pec powder and stirred at room temperature, so that Pec is adsorbed onto PLL-MoS2 by electrostatic interaction;

[0015] Step seven, after stirring is completed, unreacted Pec is removed by centrifugation, and then the remaining precipitate is redispersed in water to obtain Pec@PLL-MoS2.

[0016] Further, the molar ratio of sodium hydroxide to epsilon-polylysine hydrochloride monomer is 1:1.

[0017] Further, in step four, the stirring time is 12h, and the stirring speed is 3000rpm.

[0018] Further, in step four, the mass ratio of PLL to MoS2 nanosheet is 1:2.

[0019] Further, in step six, the mass ratio of Pec to MoS2 is 1:1.

[0020] Further, in step six, the mixing and stirring time of PLL-MoS2 and Pec powder is 4h.

[0021] The beneficial effects of the present application are:

[0022] By using the above construction method, the synergistic antibacterial nano platform Pec@PLL-MoS2 is successfully constructed, which can be stably dispersed in aqueous solution and has low cytotoxicity, can realize programmed eradication of biofilm infection through photo-thermal mediated, rapid controllable EPS enzyme hydrolysis, excellent biofilm permeability, and broad-spectrum and efficient photo-thermal sterilization performance. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0024] Figure 1 Pec@PLL-MoS2 particle size and morphology diagram of the synergistic antibacterial nano platform constructed by the present application;

[0025] Figure 2 Potential comparison diagram for the specific embodiment of the present application;

[0026] Figure 3Plate images and survival rates of S. aureus and E. coli after MoS2, PLL-MoS2 and Pec@PLL-MoS2 treatment.

[0027] Figure 4 Quantitative analysis chart of biofilm crystal violet staining treatment. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0029] In the description of the present application, it should be noted that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. For ease of description, the sizes of the various parts shown in the drawings are not drawn in accordance with the actual proportional relationship. The techniques, methods and devices known to those of ordinary skill in the relevant art can not be discussed in detail, but should be considered as part of the authorized description under appropriate circumstances. In all examples shown and discussed herein, any specific value should be interpreted as merely exemplary, and not as a limitation. Therefore, other examples of exemplary embodiments can have different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0030] Example 1:

[0031] MoS2 nanosheets (MoS2 NSs) were prepared by liquid exfoliation of bulk MoS2 in N-methyl pyrrolidone (NMP) solution. The NMP solution was removed by centrifugation and washed twice with water. The hydrochloric acid molecules in ε-polylysine hydrochloride (PLL·HCl) were removed by adding an appropriate amount of sodium hydroxide (molar ratio of sodium hydroxide: PLL·HCl monomer = 1:1). Then, MoS2 NSs and PLL were mixed at a mass ratio of 1:2 and stirred at 3000 rpm for 12 hours at room temperature to allow them to fully react. PLL was adsorbed onto MoS2 NSs by electrostatic interaction. The unreacted PLL was removed by centrifugation and washed twice with water to obtain PLL-MoS2. Finally, pectinase (Pec) powder was added to the PLL-MoS2 at a mass ratio of 1:1. After stirring at room temperature for 4 hours, Pec was adsorbed onto the surface of PLL-MoS2 by electrostatic interaction. The unreacted Pec was removed by centrifugation and washed twice with water. The precipitate was redispersed in water to obtain Pec@PLL-MoS2.

[0032] The Pec@PLL-MoS2 nanoplatform constructed in this embodiment can achieve programmed eradication of biofilm infection through photo-thermal mediated, rapid and controllable EPS enzymolysis, excellent biofilm permeability, and broad-spectrum and efficient photo-thermal sterilization performance.

[0033] As Figure 1 and Figure 2 According to the experimental results, the average size of Pec@PLL-MoS2 is 255.4 nm, and the average potential in the aqueous solution is -14.7 mV. The nanosheet with a moderate size has good photo-thermal performance (50 nm-500 nm), and the larger absolute value of the negative potential enables Pec@PLL-MoS2 to be stably dispersed in an aqueous solution and has lower cytotoxicity.

[0034] Test Example 1

[0035] Extracellular polysaccharide degradation: The mature biofilm cultured in vitro in a 96-well plate was washed twice with sterile water to remove the residual culture medium, and then 100 μL of Pec@PLL-MoS2 (200 μg / mL) was added and irradiated with 1 W of 808 nm near-infrared light for 5 minutes. After cooling to room temperature, the liquid in the well plate was aspirated, the supernatant was taken by centrifugation, and the sugar content in the solution was determined by the sulfuric acid-anthrone method to evaluate the degradation amount of extracellular polysaccharide.

[0036] Test Example 2

[0037] Antibacterial test: The mature biofilm cultured in vitro in 96-well plate was washed twice with sterile water to remove the residual culture medium, then 100 μL of Pec@PLL-MoS2 (200 μg / mL) was added and irradiated with 1 W of 808 nm near-infrared light for 5 minutes. The biofilm in the well plate was scraped off with a cell scraper, transferred to a centrifuge tube, and ultrasonicated in an ultrasonic cleaner for 2 minutes to disperse the biofilm to obtain free bacteria. Finally, the separated bacteria were cultured on TSB solid medium for 12 hours using the plate coating method, and the antibacterial rate was calculated by CFU counting method.

[0038] Test Example 3:

[0039] Antibacterial test: The mature biofilm cultured in vitro in 96-well plate was washed twice with sterile water to remove the residual culture medium, then 100 μL of Pec@PLL-MoS2 (200 μg / mL) was added and irradiated with 1 W of 808 nm near-infrared light for 5 minutes. The biofilm in the well plate was scraped off with a cell scraper, transferred to a centrifuge tube, and ultrasonicated in an ultrasonic cleaner for 2 minutes to disperse the biofilm to obtain free bacteria. Finally, the separated bacteria were cultured on TSB solid medium for 12 hours using the plate coating method, and the antibacterial rate was calculated by CFU counting method.

[0040] Pec@PLL-MoS2 can quickly degrade the extracellular polysaccharides in the bacterial biofilm matrix under the irradiation of 808 nm near-infrared light (1 W). After 5 minutes of irradiation, Pec@PLL-MoS2 can degrade 84.77% of the extracellular polysaccharides in S. aureus biofilm and 69.15% of the extracellular polysaccharides in E. coli biofilm, promoting the penetration of the material into the interior of the biofilm. Pec@PLL-MoS2 relies on its excellent photothermal properties, and after 5 minutes of irradiation, the antibacterial rates of S. aureus and E. coli in the interior of the biofilm reached 94.9% and 99.9%, respectively. In addition, the semi-quantitative analysis of biofilm crystal violet staining showed that the removal efficiency of Pec@PLL-MoS2 on S. aureus and E. coli biofilm reached 90.74% and 89.92%, respectively, after 5 minutes of irradiation.

[0041] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A synergistic antibacterial nanoplatform, characterized in that, The nano platform comprises Pec@PLL-MoS2 nanosheets composed of pectinase Pec, epsilon-polylysine PLL and MoS2 nanosheets; The method for constructing the synergistic antibacterial nano platform comprises the following steps: Step one, MoS2 nanosheets are obtained by ultrasonic liquid phase exfoliation of large-diameter MoS2 in N-methyl pyrrolidone (NMP) solution; Step two, the NMP solution is removed by centrifugation and washed with water twice; Step three, the hydrochloric acid molecules in epsilon-polylysine hydrochloride are removed by sodium hydroxide to obtain epsilon-polylysine (PLL); Step four, PLL is mixed with MoS2 nanosheets and stirred at room temperature, so that PLL is adsorbed onto MoS2 nanosheets through electrostatic interaction; Step five, the unreacted PLL in step four is removed by centrifugation and washed with water twice to obtain PLL-MoS2; Step six, the PLL-MoS2 obtained in step five is added to Pec powder and stirred at room temperature, so that Pec is adsorbed onto PLL-MoS2 through electrostatic interaction; Step seven, after stirring, the unreacted Pec is removed by centrifugation and washed with water twice, then the remaining precipitate is redispersed in water to obtain Pec@PLL-MoS2.

2. The synergistic antimicrobial nanoplatform of claim 1, wherein, In step three, the molar ratio of sodium hydroxide to epsilon-polylysine hydrochloride monomer is 1:

1.

3. The synergistic antimicrobial nanoplatform of claim 1, wherein, In step four, PLL is mixed with MoS2 nanosheets, the stirring time is 12h, and the stirring speed is 3000rpm.

4. The synergistic antimicrobial nanoplatform of claim 1, wherein, In step four, the mass ratio of PLL to MoS2 nanosheets is 1:

2.

5. The synergistic antimicrobial nanoplatform of claim 1, wherein, In step six, the mass ratio of Pec to MoS2 nanosheets is 1:

1.

6. The synergistic antimicrobial nanoplatform of claim 1, wherein, In step six, the stirring time of PLL-MoS2 and Pec powder is 4h.

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