CN111574723A

CN111574723A - Broad-spectrum antimicrobial mesoporous silica Schiff base silver complex nano material and preparation method thereof

Info

Publication number: CN111574723A
Application number: CN202010270412.2A
Authority: CN
Inventors: 陈进; 毕洪凯; 蔡铃; 黄衍强; 王建明; 刘巧
Original assignee: Jiangsu Center For Disease Control And Prevention (jiangsu Institute Of Public Health); Nanjing Medical University
Current assignee: Jiangsu Center For Disease Control And Prevention (jiangsu Institute Of Public Health); Nanjing University; Nanjing Medical University
Priority date: 2020-04-08
Filing date: 2020-04-08
Publication date: 2020-08-25
Anticipated expiration: 2040-04-08
Also published as: CN111574723B

Abstract

The invention relates to a broad-spectrum antimicrobial mesoporous silica Schiff base silver complex nano material and a preparation method thereof. The preparation method comprises the following steps: preparing aminated mesoporous silica, preparing aldehydized mesoporous silica, and preparing the finished product of the mesoporous silica Schiff base silver complex nano material. The nanometer material has the function of killing various bacteria and fungi, can achieve the effects of broad spectrum, high efficiency and continuous sterilization, and can prevent the generation of drug-resistant strains while resisting bacteria.

Description

Broad-spectrum antimicrobial mesoporous silica Schiff base silver complex nano material and preparation method thereof

Technical Field

The invention relates to a broad-spectrum antimicrobial mesoporous silica Schiff base silver complex nano material and a preparation method thereof, belonging to the technical field of antimicrobial materials.

Background

According to the knowledge of the inventor, the existing antibacterial material is complex in process during synthesis, and needs to consume a large amount of manpower, material resources and financial resources, so that the cost is high, and the market popularization is not facilitated. Meanwhile, the existing antibacterial material has limited sterilization objects and usually has a sterilization effect only on certain bacteria; the sterilization duration is short, and the long-time continuous sterilization is difficult; after a large amount of the composition is used, drug-resistant strains are easy to generate, and the health of human beings is threatened. In addition, some silver-based antibacterial materials are harmful to organisms due to high silver content, and cannot directly act on animals or human beings. Accordingly, there is a need to develop antimicrobial materials that overcome the above disadvantages.

The inventor of the invention has filed a Chinese invention patent named nanoparticle composite material, a synthetic method and application thereof on 2018, 03.01.03. the composite material SBA-15/PDA/Ag recorded in the Chinese invention patent has an antibacterial effect. After that, as a result of further research, the inventors have obtained a new research result that can overcome the above-mentioned drawbacks of the conventional materials, and have applied for the present invention.

Disclosure of Invention

The invention aims to: the problems in the prior art are solved, the preparation method of the broad-spectrum antimicrobial mesoporous silica Schiff base silver complex nano material is provided, the synthesis process is green, simple and rapid, the obtained material has a killing effect on various bacterial fungi, and broad-spectrum, efficient and continuous sterilization is realized. Meanwhile, the nano material prepared by the preparation method and the application of the nano material are also provided.

The technical scheme for solving the technical problems of the invention is as follows:

a preparation method of a broad-spectrum antimicrobial mesoporous silica Schiff base silver complex nano material is characterized by comprising the following steps:

firstly, dispersing mesoporous silicon oxide in toluene, adding 3-aminopropyltriethoxysilane, and reacting under reaction conditions; after the reaction, centrifuging and collecting solid matters; washing and drying the solid to obtain aminated mesoporous silica;

secondly, dispersing the aminated mesoporous silica in deionized water, adding glyoxylic acid, and reacting under reaction conditions; after the reaction, centrifuging and collecting solid matters; washing and drying the solid to obtain aldehyde mesoporous silica;

dispersing polylysine in absolute ethyl alcohol to form a mixed solution, and adjusting the pH value of the mixed solution to be alkalescent by using potassium hydroxide, wherein the pH value is 8.0 +/-0.5; adding aldehyde mesoporous silicon oxide to react under reaction conditions; after the reaction, adding soluble silver salt into the mixed solution, and then continuing the reaction; after the reaction is finished, centrifuging and collecting solid matters; and washing and drying the solid to obtain the polylysine modified mesoporous silica loaded with the silver nanoparticles, namely the finished product of the mesoporous silica Schiff base silver complex nano material.

The method has the advantages of green, simple and quick synthesis process, less silver required to be added, and low silver content in the prepared nano material. The nanometer material prepared by the method has a killing effect on various bacteria and fungi, can achieve broad-spectrum, high-efficiency and continuous sterilization effects, and can prevent the generation of drug-resistant strains while resisting bacteria. Compared with the composite material SBA-15/PDA/Ag invented by the prior subject group, the nano material prepared by the invention has more obvious effect of killing mycobacterium tuberculosis.

The technical scheme of the invention is further perfected as follows:

preferably, in the first step, the mass-to-volume ratio of the mesoporous silica to the toluene is 0.6 ± 0.01 g: 80 plus or minus 5 ml; the volume ratio of the 3-aminopropyltriethoxysilane to the toluene is 0.0025 +/-0.001: 1; adding 3-aminopropyltriethoxysilane drop by drop; the reaction conditions of the first step are as follows: the reaction temperature is 70 +/-5 ℃, the reaction time is at least 12 hours, and the stirring is continued in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with ethanol and deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

By adopting the preferable scheme, the material proportion and specific conditions in the first step can be further optimized. Wherein 3-aminopropyltriethoxysilane is APTES.

Preferably, in the second step, the mass-to-volume ratio of the aminated mesoporous silica to the deionized water is 0.5 ± 0.01 g: 100 plus or minus 10 ml; the molar ratio of the glyoxylic acid to the 3-aminopropyltriethoxysilane of the first step is greater than 1.

With this preferred embodiment, the proportions of the individual components in the second step can be further optimized. Wherein, the molar ratio of the glyoxylic acid to the 3-aminopropyltriethoxysilane in the first step is more than 1, so that the excess glyoxylic acid can be ensured to realize better reaction effect.

Preferably, the reaction conditions of the second step are: the reaction temperature is 40 +/-5 ℃, the reaction time is at least 6 hours, and the stirring is continued in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

With this preferred embodiment, each specific condition in the second step can be further optimized.

Preferably, in the third step, the mass-volume ratio of the polylysine to the absolute ethyl alcohol is 120 +/-20 mg: 60 plus or minus 10ml, and the molecular weight of the polylysine is 3000 plus or minus 500; the mass ratio of the aldehyde mesoporous silicon oxide to polylysine is 0.5-2: 1; the soluble silver salt is silver nitrate, and the mass ratio of the silver nitrate to the aldehyde mesoporous silicon oxide is 1.4 +/-0.2: 1.

by adopting the preferred scheme, the material proportion in the third step can be further optimized. The input amount of the soluble silver salt is obviously less than that of SBA-15/PDA/Ag, so that the silver content of the nano material prepared by the method is lower.

Preferably, the reaction conditions of the third step are: the reaction temperature is 80 +/-5 ℃, the reaction time is at least 12 hours, and condensation reflux is carried out in the reaction process; the reaction conditions for the further reaction are: the reaction temperature is 80 ℃ plus or minus 5 ℃, the reaction time is at least 30 minutes, and the condensation reflux is carried out in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with anhydrous ethanol and deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

With this preferred embodiment, each specific condition in the third step can be further optimized.

Preferably, in the first step, the mesoporous silica is SBA-15, and the preparation process comprises:

s1, adding P123 into deionized water, and stirring until the mixture is clear; adding hydrochloric acid solution and mixing uniformly; adding tetraethoxysilane and stirring for reaction;

s2, transferring the obtained mixture to a high-temperature reaction kettle for reaction, then aging the mixture in a drying oven, and washing and drying the obtained solid matter;

and S3, grinding the solid matter into powder and calcining to obtain the SBA-15.

By adopting the preferred scheme, the specific preparation process of the mesoporous silicon oxide can be further defined.

The invention also proposes:

the mesoporous silica Schiff base silver complex nano material prepared by the preparation method is adopted.

The nanometer material has the function of killing various bacteria and fungi, can achieve the effects of broad spectrum, high efficiency and continuous sterilization, and can prevent the generation of drug-resistant strains while resisting bacteria.

The invention also proposes:

the application of the mesoporous silica Schiff base silver complex nano material is characterized in that the application is used for preparing an antimicrobial agent.

The use is that the nanomaterial described hereinbefore is suitable for antimicrobial function.

Preferably, the antimicrobial agent is directed against a microorganism including Candida albicans, Candida tropicalis, Candida glabrata, Candida vitis, Candida parapsilosis, Candida ruxoides, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica subspecies, Klebsiella pneumoniae, Morganella morganii, stenotrophomonas maltophilia, Proteus mirabilis, Acinetobacter baumannii, Staphylococcus aureus, enterococcus faecalis, enterococcus faecium, Bacillus subtilis, Bacillus cereus, Moraxella catarrhalis, Streptococcus pneumoniae, Actinobacillus actinobacillus and Mycobacterium tuberculosis.

With this preferred embodiment, the kind of microorganism to be targeted can be further specified.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method is green, simple and quick, and the silver content in the prepared nano material is lower due to less silver required to be input. The nano material has a killing effect on various bacteria and fungi, can achieve the effects of broad spectrum, high efficiency and continuous sterilization, and can prevent the generation of drug-resistant strains while resisting bacteria.

Drawings

FIG. 1 shows scanning electron micrographs (A: SBA-15, B: CLA-1) and transmission electron micrographs (C: SBA-15, D: CLA-1) of example 4 of the present invention.

FIG. 2 is a graph showing the energy dispersion spectrum of CLA-1 in example 4 of the present invention.

FIG. 3 is a Fourier infrared spectrum of CLA-1 and each control group in example 4 of the present invention.

FIG. 4 shows the results of example 4 of the present invention¹³C solid nuclear magnetic resonance spectrum.

FIG. 5 is a graph showing the results of evaluating the growth effects of Escherichia coli and Staphylococcus aureus (after 72 hours of co-culture) in example 5 of the present invention.

FIG. 6 is a graph showing the results of evaluating the growth effect of Mycobacterium tuberculosis in example 5 of the present invention (after 42 days of co-culture).

Fig. 7 is a corresponding schematic diagram of embodiment 6 of the present invention.

FIG. 8 is a graph showing the results of example 7 of the present invention.

FIG. 9 is a graph showing the development results of resistance to CLA-1 and Fluconazole (FLC) by Candida albicans SC5314 of example 8 of the present invention.

FIG. 10 is a diagram of the main embodiment of the present invention.

Detailed Description

The invention is described in further detail below with reference to embodiments and with reference to the drawings. The invention is not limited to the examples given.

Example 1

This example is to prepare mesoporous silica SBA-15.

The basic preparation process of this example comprises:

s1, adding P123 into deionized water, and stirring until the mixture is clear; adding hydrochloric acid solution and mixing uniformly; adding tetraethoxysilane and stirring to react.

And S2, transferring the obtained mixture to a high-temperature reaction kettle for reaction, then aging in an oven, and washing and drying the obtained solid matter.

The following are exemplary specific preparation procedures:

weighing 4.0 g of P123, putting the P123 into a three-neck flask, adding 80 ml of deionized water, and stirring at normal temperature until the mixture is clear; measuring 70 ml of 3.4M HCl, adding the HCl into the clear solution, and stirring the solution at the constant temperature of 40 ℃ for 1 hour; under the stirring state, 9.14 ml of tetraethoxysilane is added drop by drop, and the stirring is continued at the constant temperature of 40 ℃ for 24 hours; transferring the reaction liquid to a high-temperature reaction kettle for continuous reaction, aging in an oven at 100 ℃ for 24 hours, taking solid matters, washing with water and ethanol for three times respectively, centrifuging to remove supernatant, drying, grinding to obtain powder, calcining at 550 ℃ for 6 hours, and removing a template agent to obtain the mesoporous silicon oxide SBA-15. Stored for later use in the examples.

Example 2

This example is to prepare aldehyde mesoporous silica.

The basic preparation process of this example comprises:

firstly, dispersing the mesoporous silica obtained in the example 1 in toluene, adding 3-aminopropyltriethoxysilane, and reacting under reaction conditions; after the reaction, centrifuging and collecting solid matters; and washing and drying the solid to obtain the aminated mesoporous silica. Denoted NH₂-SBA-15。

Wherein the mass volume ratio of the mesoporous silicon oxide to the toluene is 0.6 +/-0.01 g: 80 plus or minus 5 ml; the volume ratio of the 3-aminopropyltriethoxysilane to the toluene is 0.0025 +/-0.001: 1. adding 3-aminopropyltriethoxysilane drop by drop; the specific reaction conditions are as follows: the reaction temperature is 70 +/-5 ℃, the reaction time is at least 12 hours, and the stirring is continued in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with ethanol and deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

Secondly, dispersing the aminated mesoporous silica obtained in the first step in deionized water, adding glyoxylic acid, and reacting under reaction conditions; after the reaction, centrifuging and collecting solid matters; and washing and drying the solid to obtain the aldehyde mesoporous silica. Designated CHO-SBA-15.

Wherein the mass-volume ratio of the aminated mesoporous silicon oxide to the deionized water is 0.5 +/-0.01 g: 100 plus or minus 10 ml; the molar ratio of glyoxylic acid to 3-aminopropyltriethoxysilane in the first step is greater than 1. The specific reaction conditions are as follows: the reaction temperature is 40 +/-5 ℃, the reaction time is at least 6 hours, and the stirring is continued in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

The following are exemplary specific preparation procedures:

first, 0.6 g of SBA-15 of example 1 was weighed and dispersed in 80 ml of toluene, stirred uniformly and then 0.2ml of 3-Aminopropyltriethoxysilane (APTES) was added dropwise, stirred continuously in a constant temperature oil bath at 70 ℃ for 12 hours, and centrifuged at 5000rpm for 5 minutes. Removing supernatant, washing with ethanol and deionized water for 3 times, and drying at 60 deg.C to obtain aminated mesoporous silica (denoted as NH)₂-SBA-15。

Second, 0.5 g of NH obtained in the first step was weighed₂-SBA-15, dispersing in 100 ml of deionized water, adding excess glyoxylic acid (the molar ratio of the glyoxylic acid to the 3-aminopropyltriethoxysilane in the first step is more than 1 to ensure excess), stirring at constant temperature of 40 ℃ for 6 hours, centrifuging the obtained mixture for 5 minutes at 5000rpm, removing supernatant, washing with deionized water for 3 times, and drying at 60 ℃ overnight to obtain the aldehyde mesoporous silica, which is recorded as CHO-SBA-15.

Example 3

This example is to prepare polylysine-modified mesoporous silica, and polylysine-modified mesoporous silica loaded with silver nanoparticles.

The basic preparation procedure of this example is as follows:

(1) preparation of polylysine-modified mesoporous silica:

dispersing polylysine in absolute ethyl alcohol to form a mixed solution, and adjusting the pH value of the mixed solution to be alkalescent by using potassium hydroxide, wherein the pH value is 8.0 +/-0.5; adding the aldehyde mesoporous silica obtained in the example 2, and reacting under the reaction condition; after the reaction is finished, centrifuging and collecting solid matters; and washing and drying the solid to obtain the polylysine modified mesoporous silica. It is referred to as CHO-SBA-15/-PL.

(2) Preparing the silver nanoparticle-loaded polylysine modified mesoporous silica:

dispersing polylysine in absolute ethyl alcohol to form a mixed solution, and adjusting the pH value of the mixed solution to be alkalescent by using potassium hydroxide, wherein the pH value is 8.0 +/-0.5; adding the aldehyde mesoporous silica obtained in the example 2, and reacting under the reaction condition; after the reaction, adding soluble silver salt into the mixed solution, and then continuing the reaction; after the reaction is finished, centrifuging and collecting solid matters; and washing and drying the solid to obtain the polylysine modified mesoporous silica loaded with the silver nanoparticles, namely the finished product of the mesoporous silica Schiff base silver complex nano material. It is referred to as CHO-SBA-15/-PL/Ag (also referred to as CLA-1).

For the same parts of the steps in (1) and (2), the same parameters are used.

Wherein the mass-volume ratio of polylysine to absolute ethyl alcohol is 120 plus or minus 20 mg: 60 plus or minus 10ml, and the molecular weight of polylysine is 3000 plus or minus 500; the mass ratio of the aldehyde mesoporous silica to the polylysine is 0.5-2: 1; the soluble silver salt is silver nitrate, and the mass ratio of the silver nitrate to the aldehyde mesoporous silicon oxide is 1.4 +/-0.2: 1.

the specific reaction conditions are as follows: the reaction temperature is 80 +/-5 ℃, the reaction time is at least 12 hours, and condensation reflux is carried out in the reaction process; the reaction conditions for the further reaction are: the reaction temperature is 80 ℃ plus or minus 5 ℃, the reaction time is at least 30 minutes, and the condensation reflux is carried out in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with anhydrous ethanol and deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

The following are exemplary specific preparation procedures:

(1) preparation of polylysine-modified mesoporous silica:

weighing 120 mg of polylysine (-PL, the molecular weight is about 3000), dispersing into 60 ml of absolute ethyl alcohol, stirring uniformly, adding a proper amount of potassium hydroxide to make the mixture alkalescent, and enabling the pH to be 8.0 +/-0.5; 120 mg of CHO-SBA-15 from example 2 were added and the mixture was refluxed at 80 ℃ for 12 hours; the mixture was centrifuged (5 minutes at 5000 rpm), and the obtained precipitate was washed with anhydrous ethanol and deionized water for 3 times, respectively, and then dried at 60 ℃ to obtain polylysine-modified mesoporous silica, which was referred to as CHO-SBA-15/-PL, which is mesoporous silica Schiff base.

weighing 120 mg of polylysine (-PL, the molecular weight is about 3000), dispersing into 60 ml of absolute ethyl alcohol, stirring uniformly, adding a proper amount of potassium hydroxide to make the mixture alkalescent, and enabling the pH to be 8.0 +/-0.5; 120 mg of CHO-SBA-15 from example 2 were added and the mixture was refluxed at 80 ℃ for 12 hours; and then, adding 170mg of silver nitrate into the mixed solution, refluxing for 30 minutes at 80 ℃, centrifuging the mixture (5 minutes at 5000 rpm), washing the obtained precipitate for 3 times respectively by using absolute ethyl alcohol and deionized water, and drying at 60 ℃ to obtain the silver nanoparticle-loaded polylysine modified mesoporous silica, namely the mesoporous silica Schiff base silver complex nano material finished product, which is marked as CHO-SBA-15/-PL/Ag (also marked as CLA-1) and is a complex of mesoporous silica Schiff base and silver.

It should be noted that the silver nitrate charge amount in this example is 41.5% of the total mass, which can basically represent the silver nitrate charge ratio of the present invention. Compared with the SBA-15/PDA/Ag mentioned in the background art, the silver nitrate feeding proportion of the SBA-15/PDA/Ag material is basically 45.0%, the silver nitrate feeding proportion of the SBA-15/PDA/Ag material is reduced by about 7.8%, and the silver nitrate feeding proportion is obviously reduced.

Example 4

The CLA-1 prepared in example 3 was characterized, and the results thereof included a Scanning Electron Microscope (SEM) image, a Transmission Electron Microscope (TEM) image, an energy dispersion spectrogram, a BJH pore size distribution test result, a fourier infrared spectrogram, and a solid nuclear magnetic resonance spectrogram. The control group used for identification was selected from SBA-15, CHO-SBA-15/-PL.

The following are the results of the characterization of CLA-1 prepared as exemplified in example 3 (and for each control: SBA-15 prepared as exemplified in example 1, CHO-SBA-15 prepared as exemplified in example 2, CHO-SBA-15/-PL prepared as exemplified in example 3):

FIG. 1 is a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM) of SBA-15 and CLA-1, and it can be seen from the images that the SBA-15 is regular in shape and in a short rod shape, and after modification and loading of polylysine and silver particles, the regular structure of the short rod shape is not obviously changed, which indicates that the morphological characteristics of the SBA-15 are not changed in the preparation process of the composite material. As can be seen in a transmission electron microscope picture, black silver nano particles appear on the CLA-1, which indicates that silver successfully enters the pore canal of the mesoporous silicon oxide.

FIG. 2 is an energy dispersion spectrogram of CLA-1, in which it can be seen that the composite material contains elements such as C, N, O, Si, Ag, etc., and the elements Si and Ag are uniformly distributed, which proves that the silver particles are uniformly distributed in the pores of the mesoporous silica.

Table 1 reports the pore sizes of SBA-15, CHO-SBA-15/-PL, and CLA-1 obtained by BJH pore size distribution test, and it can be seen that the pore size of SBA-15 gradually decreases with the grafting of amino and aldehyde groups and loading of polylysine and silver, indicating successful grafting of aldehyde groups on SBA-15 and successful loading of each component.

TABLE 1 pore size of each sample

FIG. 3 is a Fourier infrared spectrum of CLA-1 and each control group (SBA-15, CHO-SBA-15/-PL), and CLA-1 has a new C ═ N characteristic peak at 1558 wave number, which is caused by drift after coordination of lone pair electrons of nitrogen in Schiff base and silver ions.

FIG. 4 shows CHO-SBA-15 and CHO-SBA-15/-PL¹³The C solid nuclear magnetic resonance spectrogram shows that an aldehyde characteristic peak at 173ppm disappears after polylysine is loaded, and a new C-N peak appears at 165ppm, so that the formation of a Schiff base structure (-C-N-) is further proved.

In addition, the results of characteristic identification of other CLA-1 prepared in example 3 were the same as or substantially the same as the above results, and the conclusions drawn from the results of characteristic identification were the same as the above conclusions.

Example 5

In this example, various antibacterial experiments were carried out using CHO-SBA-15/-PL and CLA-1 prepared in example 3.

The following are specific results of CHO-SBA-15/-PL, CLA-1 prepared as exemplified in example 3.

CHO-SBA-15/-PL and CLA-1 were respectively made into suspension, uniformly coated on solid culture medium, inoculated with Escherichia coli (E.coli) or Staphylococcus aureus (S.aureus) by surface coating method, and co-cultured at 37 deg.C, with the experimental results shown in FIG. 5. The results show that CHO-SBA-15/-PL did not exhibit significant antibacterial properties, whereas CLA-1 coated medium was maintained for 72 hours without growth of E.coli and S.aureus.

Different concentrations of CHO-SBA-15/-PL and CLA-1 were applied to slant culture medium and inoculated with Mycobacterium tuberculosis (M.tuberculosis), and the results after 42 days of culture are shown in FIG. 6. The results show that high concentration CLA-1 has obvious killing effect on Mycobacterium tuberculosis, and CHO-SBA-15/-PL can not inhibit the growth of Mycobacterium tuberculosis under various concentrations.

In addition, SBA-15/PDA/Ag, CLA-1 mentioned in the background art at the same concentration were co-cultured with Mycobacterium tuberculosis, and the number of colonies at different doses was observed and recorded. The results are shown in Table 2, when the dosage is 200. mu.L, both compounds show obvious killing effect on Mycobacterium tuberculosis, while the colony number of the CLA-1 treatment group is less under the action of lower dosage, which indicates that CLA-1 has obvious stronger inhibition effect on the growth of Mycobacterium tuberculosis.

TABLE 2 colony count results after co-culture of SBA-15/PDA/Ag and CLA-1 with Mycobacterium tuberculosis

The Minimal Inhibitory Concentration (MIC) of CLA-1 to various bacteria is detected by a microdilution method, the MIC is detected according to the operational standard of the CLSI antibacterial drug sensitivity test (version 2019), the experimental result is shown in Table 3, CHO-SBA-15/-PL has no antibacterial activity to various bacteria at 64 mu g/mL, and CLA-1 has the MIC of 4-32 mu g/mL to various strains, and the MIC comprises Escherichia coli, pseudomonas aeruginosa, salmonella enterica subspecies, Klebsiella pneumoniae, Morganella morganii, stenotrophomonas maltophilia, Proteus mirabilis, Acinetobacter baumannii, Staphylococcus aureus, enterococcus faecalis, enterococcus faecium, Bacillus subtilis, Bacillus cereus, Moraxella catarrhalis, Streptococcus pneumoniae and Actinobacillus actinobacillus.

TABLE 3 antibacterial Activity of CHO-SBA-15/-PL, CLA-1 against various bacteria

The in vitro bacteriostatic activity of the recombinant human anti-fungal agent on Candida albicans is evaluated by determining the Minimal Inhibitory Concentration (MIC) of CHO-SBA-15/-PL and CLA-1, and a common antifungal drug amphotericin B (AMB) is used as a positive control. Results are shown in Table 4, CHO-SBA-15/-PL showed no antibacterial activity against Candida at 64. mu.g/mL (. mu.g/mL), while CLA-1 had the same or even lower MIC for each strain than the positive control.

TABLE 4 antifungal Activity of CHO-SBA-15/-PL, CLA-1 and amphotericin B (AMB) against various Candida species

In addition, the results of the other CHO-SBA-15/-PL and CLA-1 experiments prepared in example 3 are the same or substantially the same as the above results, and the same conclusions can be drawn from the results of the experiments.

The results show that CLA-1 obviously has broad-spectrum long-acting bactericidal effect, and the microorganisms targeted by the CLA-1 comprise Candida albicans, Candida tropicalis, Candida glabrata, Candida viticola, Candida parapsilosis, Candida ruxosa, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica subspecies, Klebsiella pneumoniae, Morganella morganii, stenotrophomonas maltophilia, Proteus mirabilis, Acinetobacter baumannii, Staphylococcus aureus, enterococcus faecalis, enterococcus faecium, Bacillus subtilis, Bacillus cereus, Moraxella catarrhalis, Streptococcus pneumoniae, Actinobacillus actinomycetemcomitans and Mycobacterium tuberculosis.

Example 6

In this example, the in vivo anti-candida albicans detection was performed using CLA-1 prepared in example 3, as follows:

(1) infection and treatment time are shown in panel a of figure 7.

(2) Preparation of bacterial liquid

Inoculating the strain on a Sabouraud's agar culture medium, culturing at 37 ℃ for 48h, selecting newly cultured activated Candida albicans SC5314 or YY1-4 bacterial colonies, dissolving in a 1640 culture medium, placing on a vortex apparatus, shaking, mixing uniformly, wherein OD530 is 2-3, and the concentration of the bacterial suspension is about 2-3 × 10⁷CFU/ml, ready to use.

(3) Immunosuppressant treatment

Since immunocompetent mice are not normally colonized by candida albicans, the mice were susceptible to candidiasis by subcutaneous injection of 0.2ml hydrocortisone (200mg/kg body weight) at the back and neck of the patient 4 times a day, 1 time a day, simultaneously with infection.

(4) Inoculation of infection

Anesthetizing mouse with chloral hydrate 0.2ml (sterile water dissolved chloral hydrate, concentration of 4%), placing the animal on an electric blanket maintained at 37 deg.C, and adding Candida albicans 50 μ l (1-1.5 × 10) dropwise to the cotton ball⁶CFU/one), place cotton ball on tongue for 60-90 minutes. Animals were placed in a supine position and monitored until they recovered from anesthesia.

(5) Model evaluation

Checking the planting amount: and (3) taking the mouse tongue, crushing and coating the mouse tongue, and calculating the planting amount of each gram of tissue.

Histopathological examination: the mouse tongue was fixed in 10% formalin and examined histopathologically for HE and PAS staining, respectively.

(6) Drug delivery therapy

Setting the drug concentration and administration group, dipping the cotton balls in 80-100 mul of liquid medicine, wherein the dosage of CLA-1, Amphotericin (AMB) and Fluconazole (FLC) is 2.5mg/Kg, smearing the liquid medicine on the oral cavity and tongue of the mouse, standing for 2-3 minutes, smearing the liquid medicine for 1 time every day, and continuously taking 3 days.

(7) Treatment evaluation

Tongue was taken every other day after the 3 rd treatment for the amount of colonization examination and for HE and PAS staining pathological examination.

(8) Therapeutic results

The results are shown in B, C and D of fig. 7, and the CLA-1 treatment group is obviously superior to AMB and FLC groups, and the candida albicans colonization amount, tongue tissue inflammation and candida adhesion are reduced.

Example 7

In the embodiment, CLA-1 prepared in example 3 is adopted for toxicity detection, the drug dose is 10 times of the therapeutic dose (25mg/Kg), 80-100 mul of CLA-1 is soaked in cotton swabs, the cotton swabs are coated on the skin of a mouse, and after 30 minutes, whether rash exists or not is observed, so that the toxicity of CLA-1 on the skin is evaluated; coating the tongue of the oral cavity of the mouse, observing whether rash exists or not after 30 minutes, and evaluating the toxicity of CLA-1 on the tongue mucosa of the mouse; coating the tongue of the oral cavity of the mouse for 1 time every day for 7 days continuously, weighing the weight, and evaluating the toxicity of CLA-1 on the weight of the mouse; tongue tissues of the mice coated for 7 days are collected, pathological section HE staining observation is carried out, and the pathological toxicity of CLA-1 on the mouse tongue is evaluated. The results of the above tests are shown in fig. 8, and the results show that no rash appears on the skin (fig. 8 a) or tongue mucosa (fig. 8B), no significant change in the body weight of the mouse occurs (fig. 8C), and the HE staining results of the tongue tissue sections are normal (fig. 8D). This indicates that CLA-1 is very low in toxicity at 10 times the therapeutic dose (25mg/kg) and can be administered safely.

Example 8

In this example, the drug resistance test was carried out using CLA-1 prepared in example 3.

The following are specific results for CLA-1 prepared as exemplified in example 3.

Fluconazole (FLC) and CLA-1 were added to candida albicans suspensions at 0.5 xmic concentration, respectively, incubated at 30 ℃ for 2 days, the fungal growth was measured with a microplate reader, and the subsequent cultures were inoculated with cells. The process was repeated for 30 cycles (60 days), and the MIC changes of Fluconazole (FLC) and CLA-1 to Candida albicans SC5314 are shown in FIG. 7, which shows that the Candida albicans rapidly develops drug resistance within several days of exposure to fluconazole, while the Candida albicans is not observed to develop drug resistance during continuous passage when the sub-lethal concentration of CLA-1 exceeds 60 days.

In addition, the experimental results of other CLA-1 prepared in example 3 were the same as or substantially the same as the above results, and the conclusions drawn from the experimental results were the same as the above conclusions.

The results show that CLA-1 can prevent the generation of drug-resistant strains while resisting bacteria.

The main scheme of the invention is shown in fig. 8, and comprises the following steps:

(1) the synthetic route is as follows: SBA-15-NH₂-SBA-15——CHO-SBA-15——CHO-SBA-15/-PL——CHO-SBA-15/-PL/Ag(CLA-1)。

(2) CLA-1 has effects in killing bacteria and fungi, and preventing generation of drug-resistant strain.

In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Claims

1. A preparation method of a broad-spectrum antimicrobial mesoporous silica Schiff base silver complex nano material is characterized by comprising the following steps:

2. The method according to claim 1, wherein in the first step, the mass-to-volume ratio of the mesoporous silica to toluene is 0.6 ± 0.01 g: 80 plus or minus 5 ml; the volume ratio of the 3-aminopropyltriethoxysilane to the toluene is 0.0025 +/-0.001: 1; adding 3-aminopropyltriethoxysilane drop by drop; the reaction conditions of the first step are as follows: the reaction temperature is 70 +/-5 ℃, the reaction time is at least 12 hours, and the stirring is continued in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with ethanol and deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

3. The preparation method of claim 1, wherein in the second step, the mass-to-volume ratio of the aminated mesoporous silica to the deionized water is 0.5 ± 0.01 g: 100 plus or minus 10 ml; the molar ratio of the glyoxylic acid to the 3-aminopropyltriethoxysilane of the first step is greater than 1.

4. The process according to claim 3, wherein the reaction conditions in the second step are as follows: the reaction temperature is 40 +/-5 ℃, the reaction time is at least 6 hours, and the stirring is continued in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

5. The method according to claim 1, wherein in the third step, the mass-to-volume ratio of polylysine to absolute ethanol is 120 ± 20 mg: 60 plus or minus 10ml, and the molecular weight of the polylysine is 3000 plus or minus 500; the mass ratio of the aldehyde mesoporous silicon oxide to polylysine is 0.5-2: 1; the soluble silver salt is silver nitrate, and the mass ratio of the silver nitrate to the aldehyde mesoporous silicon oxide is 1.4 +/-0.2: 1.

6. the method according to claim 5, wherein the reaction conditions in the third step are as follows: the reaction temperature is 80 +/-5 ℃, the reaction time is at least 12 hours, and condensation reflux is carried out in the reaction process; the reaction conditions for the further reaction are: the reaction temperature is 80 ℃ plus or minus 5 ℃, the reaction time is at least 30 minutes, and the condensation reflux is carried out in the reaction process; the centrifugation conditions were: the centrifugal speed is 5000 plus or minus 500rpm, and the centrifugal time is at least 5 minutes; the washing conditions were: washing with anhydrous ethanol and deionized water for at least 3 times; the drying temperature was 60 ℃. + -. 5 ℃.

7. The method according to claim 1, wherein the mesoporous silica is SBA-15 and is prepared by the following steps:

8. The mesoporous silica Schiff base silver complex nano-material prepared by the preparation method of any one of claims 1 to 7.

9. Use of the mesoporous silica schiff base silver complex nanomaterial of claim 8, wherein the use is for preparing an antimicrobial agent.

10. The use according to claim 9, wherein the antimicrobial agent is directed against a microorganism comprising candida albicans, candida tropicalis, candida glabrata, candida vitis, candida parapsilosis, candida luxata, escherichia coli, pseudomonas aeruginosa, salmonella enterica subspecies, klebsiella pneumoniae, morganella morganii, stenotrophomonas maltophilia, proteus mirabilis, acinetobacter baumannii, staphylococcus aureus, enterococcus faecalis, enterococcus faecium, bacillus subtilis, bacillus cereus, moraxella catarrhalis, streptococcus pneumoniae, actinobacillus and mycobacterium tuberculosis.