CN112056324B - Autocatalytic antibacterial agent of polysaccharide-based nanodot aggregate and preparation method and application thereof - Google Patents
Autocatalytic antibacterial agent of polysaccharide-based nanodot aggregate and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
- A01N59/20—Copper
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
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Abstract
The invention discloses an autocatalytic antibacterial agent of a polysaccharide-based nanodot aggregate and a preparation method and application thereof, belongs to the technical field of antibacterial nanometer, and relates to an antibacterial agent and a preparation method and application thereof. The invention aims to solve the problems of poor antibacterial effect and large side effect of the existing nano antibacterial agent due to low permeability of the existing nano antibacterial agent to a bacterial biofilm. Autocatalytic antibacterial agent of polysaccharide-based nano-dot aggregate releases Cu under acidic condition2+And H2O2The Fenton reaction generates catalytically active OH, and Cu is not released under neutral or alkaline conditions2+And H2O2. The method comprises the following steps: firstly, preparing glucan/CuCl2A solution; secondly, adding NaOH solution and H2O2Stirring the solution for reaction; thirdly, centrifugal cleaning. An autocatalytic antimicrobial agent of polysaccharide-based nanodot aggregates releases catalytically active-OH under acidic conditions for killing bacteria, inhibiting the formation of bacterial biofilms, and removing formed biofilms.
Description
Technical Field
The invention belongs to the field of antibacterial nanotechnology, and relates to an antibacterial agent, and a preparation method and application thereof.
Background
About 70 million patients die each year worldwide from pathogenic infections, which makes pathogenic bacterial infections the second leading cause of death worldwide and raise widespread health concerns. Among them, the formation of biofilm is important in most bacterial infectious diseases. Biofilms are complex multicellular aggregates in which microbial cells are embedded in an autogenous matrix of extracellular polymers, including proteins, polysaccharides, extracellular nucleic acids (eDNA) and cell debris, often present on tissue surfaces and in medical devices such as catheters, pacemakers and artificial joints, causing chronic infections. The particular structure of the biofilm provides a protective barrier to the bacteria inside, as it can protect not only the bacteria within the biofilm from attack by the host's innate immune cells, but also penetration and subsequent action of antimicrobial agents. Due to the hindrance of the biofilm, it is difficult for ordinary antibacterial agents to achieve the intended antibacterial effect, which increases both the medical burden and the economic burden.
In recent years, the fenton reaction of nanoparticles has attracted a wide interest for use in the treatment of bacterial infections because it is considered to be one of the most efficient advanced oxidation processes and has a very high broad spectrum of antibacterial activity. In the course of the Fenton reaction, Fe2+，Cu2+And Mn2+Divalent metal ions and hydrogen peroxide (H)2O2) The product, OH, has excellent oxidizing ability, which in turn produces non-selective attack on the bacteria on lipid peroxidation and disruption of bacterial membrane integrity. However, the special barrier effect of the biofilm prevents OH from exerting its maximum effect, and if the antibacterial and anti-biofilm effects are enhanced by increasing the amount of the antibacterial agent used, it may have side effects on the surrounding normal cell tissues. Therefore, there is an urgent need to develop efficient, low-toxicity, low-cost nano-antibacterial agents for use in the treatment of persistent bacterial biofilm infections.
Disclosure of Invention
The invention aims to solve the problems of poor antibacterial effect and large side effect of the existing nano antibacterial agent caused by low permeability of a bacterial biomembrane, and provides a pH-responsive glucan-modified copper peroxide nanodot aggregate antibacterial agent and a preparation method and application thereof.
Autocatalytic antibacterial agent of polysaccharide-based nano-dot aggregate releases Cu under acidic condition2+And H2O2The Fenton reaction generates catalytically active OH, and Cu is not released under neutral or alkaline conditions2+And H2O2(ii) a The autocatalytic antimicrobial agent of the polysaccharide-based nano-dot aggregate consists of CuCl2、H2O2NaOH and dextran as raw materialsIs prepared.
Further, the pH value of the acidic condition is 5.6;
further, the CuCl2The molar ratio to NaOH was 1: 1.
The preparation method of the autocatalytic antibacterial agent of the polysaccharide-based nanodot aggregate is completed according to the following steps:
first, dextran is added to CuCl2Stirring the solution until the glucan is completely dissolved to obtain glucan/CuCl2A solution;
di, di-dextran/CuCl2Adding NaOH solution into the solution, and then adding H2O2Stirring the solution for reaction to obtain a copper peroxide nanodot aggregate solution containing glucan modification;
dextran/CuCl as described in step two2CuCl in solution2The molar ratio of the NaOH solution to NaOH in the NaOH solution is 1: 1;
and thirdly, taking absolute ethyl alcohol as a cleaning agent, and centrifugally cleaning the dextran-modified copper peroxide nanodot aggregate-containing solution to obtain the autocatalysis antibacterial agent solution of the polysaccharide-based nanodot aggregate.
Further, the mass of dextran and CuCl in the step one2The volume ratio of the solution (0.2 g-0.8 g) is 5 mL;
further, the mass of dextran and CuCl in the step one2The volume ratio of the solution is 0.5g to 5 mL;
further, the CuCl in the step one2The concentration of the solution is 0.005 mol/L-0.015 mol/L.
Further, the CuCl in the step one2The concentration of the solution was 0.01 mol/L.
Further, the concentration of the NaOH solution in the step two is 0.005-0.015 mol/L;
further, the CuCl in the step one2The concentration of the solution is 0.01 mol/L;
further, H in the second step2O2The mass fraction of the solution is 28-30%;
further, the NaOH solution and H in the step two2O2The volume ratio of the solution is 5mL (80-120 muL);
further, the NaOH solution and H in the step two2O2The volume ratio of the solution is 5mL to 100 mu L;
further, the stirring reaction time in the second step is 30 min.
Further, the molecular weight of the glucan in the first step is 20000 kDa.
Furthermore, the centrifugal cleaning times in the third step are 3-5 times, the speed of each centrifugal cleaning is 8000r/min, and the time of each centrifugal cleaning is 5 min.
Further, an autocatalytic antimicrobial agent of polysaccharide-based nanodot aggregates releases OH having catalytic activity under acidic conditions for killing bacteria, inhibiting the formation of bacterial biofilm, and removing the formed biofilm.
Further, the bacteria are gram-positive bacteria or gram-negative bacteria.
Further, when the gram-positive bacteria are staphylococcus aureus, the concentration of the autocatalytic antibacterial agent of the polysaccharide-based nanodot aggregate is 0.4 mug/mL, and the sterilization rate of the polysaccharide-based nanodot aggregate to the staphylococcus aureus is 98%; when the gram-negative bacteria are salmonella typhimurium, the concentration of the autocatalytic antibacterial agent of the polysaccharide-based nanodot aggregate is 4 mug/mL, and the bactericidal rate to salmonella typhimurium is 72%.
The principle of the invention is as follows:
the invention prepares acid-induced H2O2Dextran-coated copper peroxide nanodot aggregates (hereinafter, referred to as dcponas) were self-supplied. Under the acidic environment consistent with the microenvironment of the biological membrane, DCPANAs can be decomposed into Cu2+And H2O2Subsequent triggering of Cu2+And H2O2The Fenton reaction between them produces OH which destroys bacteria and biofilms. The introduction of dextran not only facilitates penetration into the biofilm, but also improves the biocompatibility of DCPND. In addition, self-sufficient H2O2Will avoid the adsorptivity H2O2Adverse effects on normal cells, and in a neutral environment consistent with healthy tissue, DCPANAs release little Cu2+And H2O2The mechanism of acidity induction can also mitigate damage to surrounding healthy tissue.
By adopting the technical scheme of the invention, the invention has the following beneficial effects:
(1) the synthesis method is simple, the pH-responsive glucan-modified copper peroxide nanodot aggregate antibacterial agent is synthesized in one step, the operation is simple and convenient, the synthesis time is short, and the cost is low;
(2) the pH-responsive glucan-modified copper peroxide nanodot aggregate antibacterial agent synthesized by the method can release OH with strong catalytic activity in an acidic environment and can catalyze the color development of a substrate TMB;
(3) the pH-responsive glucan-modified copper peroxide nanodot aggregate antibacterial agent synthesized by the method has a good antibacterial and anti-biofilm effect;
(4) the pH-responsive glucan-modified copper peroxide nanodot aggregate antibacterial agent synthesized by the invention has a broad antibacterial and anti-biofilm effect, and can play a role in common bacteria and biological membranes thereof due to the non-selective cytotoxicity of OH;
(5) the pH-responsive glucan modified copper peroxide nanodot aggregate antibacterial agent synthesized by the method can release H in an acidic environment2O2Self-supply H2O2Reduce the externally added H2O2The resulting unwanted toxicity;
(6) the pH-responsive glucan-modified copper peroxide nanodot aggregate antibacterial agent synthesized by the method has good biocompatibility and the characteristics of acidic environment response, so that the harm of the synthesized material to healthy cell tissues is reduced; meanwhile, the biocompatibility of the material is improved by modifying the glucan;
(7) the synthesized pH-responsive glucan modified copper peroxide nanodot aggregate antibacterial agent has the pH value of 5.6Is only 0.4. mu.g mL-1The sterilization rate of staphylococcus aureus can reach 98 percent, and the concentration is 4 mug mL-1The sterilization rate of the salmonella can reach 72 percent.
Compared with the traditional antibacterial agent, the pH-responsive glucan-modified copper peroxide nanodot aggregate antibacterial agent synthesized by the invention has good antibacterial performance, and the affinity of the material to a bacterial biofilm is increased by modifying glucan, so that the anti-biofilm performance of the material is improved. In addition, the toxicity of the material is reduced due to the characteristics of controllable pH and antibiosis, the synthetic method is simple, short in synthetic time, low in cost, good in performance, strong in biological activity, good in antibacterial and anti-biofilm performance, the problem that a bacterial biofilm is difficult to remove is solved, the method is novel, and the application prospect is good.
The present invention makes available a pH-responsive antimicrobial agent.
Drawings
FIG. 1 is a schematic diagram of the principle of an autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates antimicrobial and its biofilm prepared according to one embodiment;
FIG. 2 is a transmission electron microscope image of the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
FIG. 3 is a graph showing the variation of absorbance with time for solutions of DCPANAs of different concentrations;
FIG. 4 is a graph of absorbance versus time for DCPANAs solutions when the pH of the environment is different;
FIG. 5 is a graph showing the bactericidal effect of the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates on Salmonella typhimurium obtained in step three of the example;
FIG. 6 is a graph showing the bactericidal effect of the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates on Staphylococcus aureus obtained in step three of the example;
FIG. 7 is a bar graph of the inhibition of the biofilm of Salmonella typhimurium by the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
FIG. 8 is a bar graph of the inhibition of Staphylococcus aureus biofilm by the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
FIG. 9 is a bar graph of the disruption of the formed Salmonella typhimurium biofilm and Staphylococcus aureus biofilm by the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
FIG. 10 is a photograph showing a blank set of disruptions to a formed Salmonella typhimurium biofilm;
FIG. 11 is a photograph of a disruption of a formed Salmonella typhimurium biofilm by an autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
FIG. 12 is a photograph showing the destruction of a formed Staphylococcus aureus biofilm by a blank set;
fig. 13 is a photograph showing the disruption of the formed staphylococcus aureus biofilm by the autocatalytic antimicrobial solution of the polysaccharide-based nanodot aggregate obtained in step three of the example.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit of the invention.
The first embodiment is as follows: the embodiment will be described with reference to fig. 1, and the embodiment is that the autocatalytic antimicrobial agent of the polysaccharide-based nano-dot aggregate releases Cu under acidic conditions2+And H2O2The Fenton reaction generates catalytically active OH, and Cu is not released under neutral or alkaline conditions2+And H2O2(ii) a The autocatalytic antimicrobial agent of the polysaccharide-based nano-dot aggregate consists of CuCl2、H2O2NaOH and glucan as raw materials.
The second embodiment is as follows: the present embodiment differs from the present embodiment in that: the pH value of the acidic condition is 5.6; the CuCl2The molar ratio to NaOH was 1: 1. Other steps are the same as in the first embodiment.
The third concrete implementation mode: the embodiment is a preparation method of the autocatalytic antimicrobial agent of the polysaccharide-based nanodot aggregate, which is completed by the following steps:
first, dextran is added to CuCl2Stirring the solution until the glucan is completely dissolved to obtain glucan/CuCl2A solution;
di, di-dextran/CuCl2Adding NaOH solution into the solution, and then adding H2O2Stirring the solution for reaction to obtain a copper peroxide nanodot aggregate solution containing glucan modification;
dextran/CuCl as described in step two2CuCl in solution2The molar ratio of the NaOH solution to NaOH in the NaOH solution is 1: 1;
and thirdly, taking absolute ethyl alcohol as a cleaning agent, and centrifugally cleaning the dextran-modified copper peroxide nanodot aggregate-containing solution to obtain the autocatalysis antibacterial agent solution of the polysaccharide-based nanodot aggregate.
The fourth concrete implementation mode: the present embodiment is different from the third embodiment in that: the mass of dextran and CuCl in step one2The volume ratio of the solution (0.2 g-0.8 g) is 5 mL; the CuCl mentioned in the step one2The concentration of the solution is 0.005 mol/L-0.015 mol/L. The other steps are the same as those in the third embodiment.
The fifth concrete implementation mode: the third to fourth embodiments are different from the first to fourth embodiments in that: the concentration of the NaOH solution in the step two is 0.005-0.015 mol/L; h in step two2O2The mass fraction of the solution is 28-30%; NaOH solution and H in the second step2O2The volume ratio of the solution is 5mL (80-120 muL); the stirring reaction time in the second step is 30 min. The other steps are the same as those in the third to fourth embodiments.
The sixth specific implementation mode: the third to fifth embodiments are different from the first to fifth embodiments in that: the molecular weight of the glucan in the first step is 20000 kDa. The other steps are the same as those in the third to fifth embodiments.
The seventh embodiment: the third to sixth differences from the present embodiment are as follows: the centrifugal cleaning times in the third step are 3-5 times, the speed of each centrifugal cleaning is 8000r/min, and the time of each centrifugal cleaning is 5 min. The other steps are the same as those of the third to sixth embodiments.
The specific implementation mode is eight: the embodiment is that the autocatalytic antimicrobial agent of the polysaccharide-based nanodot aggregate releases OH with catalytic activity under acidic conditions, and is used for killing bacteria, inhibiting the formation of bacterial biofilms and removing the formed biofilms.
The specific implementation method nine: the present embodiment is different from the eighth embodiment in that: the bacteria are gram-positive bacteria or gram-negative bacteria. The other steps are the same as those in embodiment eight.
The detailed implementation mode is ten: the eighth embodiment differs from the ninth embodiment in that: when the gram-positive bacteria are staphylococcus aureus, the concentration of the autocatalytic antibacterial agent of the polysaccharide-based nanodot aggregate is 0.4 mug/mL, and the sterilization rate of the autocatalytic antibacterial agent to the staphylococcus aureus is 98%; when the gram-negative bacteria are salmonella typhimurium, the concentration of the autocatalytic antibacterial agent of the polysaccharide-based nanodot aggregate is 4 mug/mL, and the bactericidal rate to salmonella typhimurium is 72%. The other steps are the same as those in the eighth to ninth embodiments.
The present invention will be described in detail below with reference to the accompanying drawings and examples.
Example 1: the preparation method of the autocatalytic antibacterial agent of the polysaccharide-based nanodot aggregate is completed according to the following steps:
firstly, 0.5g of dextran is added into 5mL of CuCl with the concentration of 0.01mol/L2Stirring the solution until the glucan is completely dissolved to obtain glucan/CuCl2A solution;
the molecular weight of the glucan in the first step is 20000 kDa;
di, di-dextran/CuCl25mL of 0.01mol/L NaOH solution was added to the solution, and 100. mu.L of 30% H was added2O2Magnetically stirring the solution for 30min to obtain dextran-modified copper peroxideA compound nanodot aggregate solution;
and thirdly, carrying out centrifugal cleaning on the solution containing the glucan-modified copper peroxide nanodot aggregate for 3 times by taking absolute ethyl alcohol as a cleaning agent, wherein the speed of each centrifugal cleaning is 8000r/min, and the time of each centrifugal cleaning is 5min, so as to obtain the self-catalytic antimicrobial agent (DCPANAs) of the polysaccharide-based nanodot aggregate.
Example the concentration of the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the procedure was determined by flame atomic absorption spectrometry and reported as 98. mu.g mL of copper-1。
FIG. 2 is a transmission electron microscope image of the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
as can be seen from fig. 2, the particle size of the copper peroxide nanodots is about 10nm, and the dextran wraps the nanodots to form aggregates of about 100 nm.
Example 2: example determination of catalytic performance of autocatalytic Antimicrobial Solutions (DCPNAs) of polysaccharide-based nanodot aggregates obtained in step three of the example:
preparing autocatalysis Antibacterial Agent Solution (DCPANAs) of the polysaccharide-based nanodot aggregate obtained in the third step into different material solutions, wherein the solvent is sterile water; the concentration of DCPANAs in the 5 material solutions is 0. mu.g/mL, 0.125. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL and 1. mu.g/mL in this order. Adding 10 μ L of TMB solution with concentration of 20mmol/L into each group of material solution, and measuring absorbance of reaction system at 625nm in each hole per minute within half an hour by using microplate reader, as shown in FIG. 3;
FIG. 3 is a graph showing the variation of absorbance with time for solutions of DCPANAs of different concentrations;
as can be seen from FIG. 3, the absorbance was positively correlated with time, and the catalytic activity was improved as the concentration of DCPANAs increased.
Taking 7 groups of material solutions with the same volume and DCPANAs with the concentrations of 0, 0.125, 0.25 and 0.5 mu g/mL respectively, adding 180 mu L of buffer solutions with different pH values into the material solutions with the concentrations of 0, 0.125, 0.25 and 0.5 mu g/mL respectively of the DCPANAs of the 7 groups, leading the pH values of the material solutions of the 7 groups to be 4.5, 5.0, 5.6, 6.5, 7.4 and 8.0 respectively, and measuring the absorbance of the reaction system at 625nm in each hole per minute by using a microplate reader within half an hour, wherein the absorbance is shown in figure 4;
FIG. 4 is a graph of absorbance versus time for DCPANAs solutions when the pH of the environment is different;
as can be seen from fig. 4, the material exhibited the best catalytic activity when the pH of the environment was 5.6, which is far better than neutral or basic environment, thus indicating that the acidic environment is indeed a key factor for the generation of OH from DCPNAs.
Example 3: bacterial culture and preparation of bacterial suspension:
firstly, bacterial culture:
in the present invention, the gram-positive bacteria staphylococcus aureus (ATCC25923) and the gram-negative bacteria salmonella typhimurium (S8xc001a) were selected as model bacteria having biofilm-forming ability to evaluate the antibacterial and anti-biofilm properties of the material. First, the bacteria were cultured overnight in an incubator at 37 ℃ on LB agar plates using the plate streaking technique, and then single colonies of Staphylococcus aureus and Salmonella typhimurium were picked out on the cultured plates, and then inoculated in 50mL of LB broth, respectively, and incubated at 37 ℃ for 12 hours with shaking.
② preparing bacterial suspension: transferring 30mL of each of the two bacteria-cultured broths from the previous step into a sterile centrifuge tube, centrifuging at 4 ℃ at 6000rpm for 5min, discarding the supernatant broth, adding the same volume of PBS buffer (1X), resuspending, centrifuging again, washing repeatedly for 3 times, finally diluting the washed bacterial suspension with PBS buffer so that the absorbance at 600nm is 0.5, and storing the prepared bacterial suspension to 4 ℃ for later use.
Example 4: determination of antibacterial Properties of Autocatalytic Antimicrobial Solutions (DCPANAs) of polysaccharide-based nanodot aggregates obtained in step three of the examples:
a plate colony counting method is adopted for evaluating the antibacterial activity of the material in different pH environments; 100. mu.L of bacterial suspension (10)5CFU/mL) was added to 890. mu.L of PBS buffer at pH 5.6, 6.5 and 7.4, respectively, to form a bacterial suspension; then adding 10 μ L of DCPANAs with different concentrations to dissolveFinally, the concentration of DCPANAs in the gram-positive bacteria sterilization system is respectively 0.1, 0.2, 0.3 and 0.4 mug/mL, and the concentration of DCPANAs in the gram-positive bacteria sterilization system is respectively 1, 2, 3 and 4 mug/mL; the blank group was replaced with a buffer of the corresponding pH for the material solution. Performing shaking incubation at 37 ℃ for 30 minutes, then coating the bacterial suspension on an LB agar plate by adopting a plate coating method, culturing at 37 ℃ for 24 hours, and calculating the survival rate of bacteria by colony count after the culture is finished, as shown in figure 5 and figure 6;
FIG. 5 is a graph showing the bactericidal effect of the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates on Salmonella typhimurium obtained in step three of the example;
FIG. 6 is a graph showing the bactericidal effect of the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates on Staphylococcus aureus obtained in step three of the example;
as can be seen from FIGS. 5 and 6, the survival rates of both Salmonella typhimurium and Staphylococcus aureus decreased with increasing material concentration. Meanwhile, the result shows that the DCPANAs have excellent antibacterial activity. Furthermore, it is noteworthy that the results of fig. 5 and 6 demonstrate that the percent survival of salmonella typhimurium and staphylococcus aureus decreases with decreasing environmental pH. The concentration was 4. mu.g mL-1The survival of salmonella typhimurium treated with DCPNAs at pH 5.6 was as low as 20% and 75.5% at pH7.4, indicating the pH-dependent antibacterial activity of DCPNAs. The bactericidal effect of Staphylococcus aureus in FIG. 6 is similar to the bactericidal effect of Salmonella typhimurium in FIG. 5 in pH responsiveness, and the concentration is 0.4. mu.g mL-1The survival rate of salmonella typhimurium treated with DCPNAs at pH 5.6 was as low as 2%.
Example 5: example biofilm resistance performance of autocatalytic antimicrobial solutions of polysaccharide-based nanodot aggregates obtained in step three:
(1) the crystal violet staining assay was used to assess the ability to inhibit biofilm formation:
80 μ L of three sets of Tryptone Soy Broth (TSB) pH 5.6, 6.5 and 7.4 were added to each well of a sterile 96 well cell culture plate, two columns per set. Then 10. mu.L of the bacterial suspension (10)8CFU/mL) was added to each groupIn a broth; then, 10. mu.L of the material solution was added to one column of each group at a concentration of 9.8. mu.g/mL; blank groups replaced material solutions with equal volumes of the corresponding TSB broth and were added to the other column of each group. Then incubated at 37 ℃ for 24h to obtain a mature biofilm; rinse with sterile saline, dry for 20 minutes, add 100 μ Ι _ crystal violet solution (0.1%, w/v) to each well to stain for 5 minutes; after washing with sterile water to remove excess dye, the biofilm stained in the wells was dissolved in acetic acid solution (33%, v/v), and finally absorbance was measured at 592nm using a microplate reader, and the biofilm quality was represented by the absorbance, as shown in FIG. 7;
FIG. 7 is a bar graph of the inhibition of the biofilm of Salmonella typhimurium by the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
FIG. 8 is a bar graph of the inhibition of Staphylococcus aureus biofilm by the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
as a result: as shown in FIG. 7, the biofilm mass of Salmonella typhimurium in the control group was about 1.8, indicating successful biofilm formation. After incubation with DCPNAs at ph7.4, the biofilm quality was similar to the control group, indicating that the material was not able to inhibit biofilm formation at ph 7.4. While a slight biofilm inhibition was observed after incubation of DCPANAs at pH 6.5. In sharp contrast, the biofilm quality in the experimental group with pH 5.6 was much lower than that in the control group, which indicates that the DCPANAs synthesized by the invention have excellent acid-induced anti-biofilm performance and can inhibit the formation of the biofilm by the Salmonella typhimurium.
As shown in FIG. 8, DCPANAs had the same effect on the inhibition of Staphylococcus aureus biofilm formation as that of FIG. 7, and showed significant inhibition under acidic conditions (pH 5.6).
(2) MTT staining assay to assess the destructive effect of materials on biofilms:
according to the method for culturing the biological membrane in the previous step, the formed biological membrane is cultured by the autocatalysis antibacterial agent solution of the polysaccharide-based nanodot aggregate obtained in the third step without the embodiment; the cultured biofilm was gently washed with sterile saline, then 10. mu.L of the LDCPNAs solution and 90. mu.L of sterile physiological saline at a material concentration of 9.8. mu.g/m were added, and incubated at 37 ℃ for 24 hours. The blank group replaced the material solution with an equal volume of sterile saline. After washing with sterile water, 100. mu.L of MTT (0.5mg/mL) solution was added to each well and incubated at 37 ℃ for 3h, then, 100. mu.L of dimethyl sulfoxide (DMSO) was further added to dissolve the bluish-purple precipitate, and finally, the absorbance of each well was measured by a microplate reader at 592 nm.
FIG. 9 is a bar graph of the disruption of the formed Salmonella typhimurium biofilm and Staphylococcus aureus biofilm by the autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
as a result: as shown in FIG. 9, after incubation with DCPANAs, the quality of the biofilm of Salmonella typhimurium and Staphylococcus aureus was significantly reduced, demonstrating the destructive effect of the synthesized DCPANAs antimicrobials of the present invention on the formed biofilm.
FIG. 10 is a photograph showing a blank set of disruptions to a formed Salmonella typhimurium biofilm;
FIG. 11 is a photograph of a disruption of a formed Salmonella typhimurium biofilm by an autocatalytic antimicrobial solution of polysaccharide-based nanodot aggregates obtained in step three of the example;
FIG. 12 is a photograph showing the destruction of a formed Staphylococcus aureus biofilm by a blank set;
fig. 13 is a photograph showing the disruption of the formed staphylococcus aureus biofilm by the autocatalytic antimicrobial solution of the polysaccharide-based nanodot aggregate obtained in step three of the example.
The phenomenon that the mature biofilm treated with the material was destroyed was recorded from the photographs in fig. 10 to 13, and the excellent biofilm removing ability of the present invention was visually demonstrated.
Claims (1)
1. The application of the polysaccharide-based nano-dot aggregate in preparing the autocatalytic antibacterial agent is characterized in that the preparation method of the autocatalytic antibacterial agent is completed according to the following steps:
firstly, 0.5g of dextran is added into 5mL of CuCl with the concentration of 0.01mol/L2Stirring the solution until the glucan is completely dissolved to obtain glucan/CuCl2A solution;
the molecular weight of the glucan in the first step is 20000 kDa;
di, di-dextran/CuCl25mL of 0.01mol/L NaOH solution was added to the solution, and 100. mu.L of 30% H was added2O2Magnetically stirring the solution for 30min to obtain a copper peroxide nanodot aggregate solution containing glucan modification;
and thirdly, carrying out centrifugal cleaning on the dextran-modified copper peroxide nanodot aggregate solution for 3 times by taking absolute ethyl alcohol as a cleaning agent, wherein the speed of each centrifugal cleaning is 8000r/min, and the time of each centrifugal cleaning is 5min, so as to obtain the autocatalytic antibacterial agent.
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