阅读说明：本技术 一种抗菌纳米材料及其制备方法和应用 (Antibacterial nano material and preparation method and application thereof ) 是由 蒋兴宇 王乐 于 2020-06-01 设计创作，主要内容包括：本发明提供一种抗菌纳米材料及其制备方法和应用,所述抗菌纳米材料为氨基苯酚修饰的金纳米颗粒,所述氨基苯酚通过金-氨键修饰在所述金纳米颗粒表面。所述氨基苯酚与金纳米颗粒结合之后,所得抗菌纳米材料表面的羟基能够靶向细菌表面的多糖,通过破坏细菌细胞壁进而增加细胞膜的通透性,并且所述氨基苯酚作为功能基团与细菌16S核糖体RNA结合,抑制蛋白质合成,进而杀灭细菌,达到较好的抗菌效果。同时,所述抗菌纳米材料表面带负电,具有优异的生物相容性,且不会诱发细菌产生耐药性,与氨基糖苷类药物等肾毒性药物相比,还具有较高的生物安全性。(The invention provides an antibacterial nano material and a preparation method and application thereof, wherein the antibacterial nano material is gold nanoparticles modified by aminophenol, and the aminophenol is modified on the surfaces of the gold nanoparticles through gold-ammonia bonds. After the aminophenol is combined with the gold nanoparticles, the obtained hydroxyl on the surface of the antibacterial nano material can target the polysaccharide on the surface of bacteria, the permeability of cell membranes is increased by destroying the cell walls of the bacteria, and the aminophenol is combined with the 16S ribosomal RNA of the bacteria as a functional group to inhibit protein synthesis, further kill the bacteria, and achieve a better antibacterial effect. Meanwhile, the antibacterial nano material has the advantages of negative charge on the surface, excellent biocompatibility, no induction of drug resistance of bacteria, and higher biological safety compared with nephrotoxic drugs such as aminoglycoside drugs and the like.)

1. The antibacterial nano material is characterized in that the antibacterial nano material is gold nanoparticles modified by aminophenol, and the aminophenol is modified on the surfaces of the gold nanoparticles through gold-ammonia bonds.

2. The antimicrobial nanomaterial of claim 1, wherein the aminophenol comprises any one or a combination of at least two of ortho-aminophenol, para-aminophenol, or meta-aminophenol;

preferably, the gold nanoparticles are prepared by adopting a sodium borohydride reduction method;

preferably, the particle size of the antibacterial nano material is 2-8 nm.

3. A method for preparing the antibacterial nanomaterial of claim 1 or 2, comprising the steps of: and mixing aminophenol with a reaction solution for preparing the gold nanoparticles, and reacting to obtain the antibacterial nano material.

4. The method for preparing antibacterial nano-material according to claim 3, characterized by comprising the following steps:

(1) mixing chloroauric acid, aminophenol, a pH regulator, a stabilizer and water, and dissolving to obtain a reaction solution;

(2) and (2) adding sodium borohydride into the reaction solution obtained in the step (1) to react, dialyzing, and sterilizing to obtain the antibacterial nano material.

5. The method according to claim 4, wherein the molar concentration of chloroauric acid in step (1) is 1 to 10 mM;

preferably, the molar concentration of the aminophenol in the step (1) is 1 to 10 mM;

preferably, the pH adjusting agent of step (1) comprises triethylamine;

preferably, the final concentration of the triethylamine is 0.1-0.15%;

preferably, the stabilizing agent of step (1) comprises tween 80;

preferably, the final concentration of tween 80 in the step (1) is 0.5 to 1 g/L;

preferably, the temperature during the dissolving in the step (1) is-5 ℃, and preferably 0 ℃.

6. The method according to claim 4 or 5, wherein the sodium borohydride in step (2) is dissolved in water and then added to the reaction solution in step (1) under stirring;

preferably, the concentration of the sodium borohydride dissolved in water is 2-5 g/L;

preferably, the rotation speed during stirring is 800-.

7. The process according to any one of claims 4 to 6, wherein the reaction time in the step (2) is 1.5 to 3 hours;

preferably, the temperature of the reaction in the step (2) is-5 ℃, and preferably 0 ℃.

8. The method according to any one of claims 4 to 7, wherein the dialysis is carried out using a dialysis bag in the step (2);

preferably, the dialysis bag has a molecular weight cut-off of 12-15 kDa;

preferably, the dialysis time of the step (2) is 20-30 h;

preferably, the sterilization method in step (2) is filtration sterilization using a 0.22 μm filter.

9. The method for preparing according to claims 4-8, characterized in that it comprises the steps of:

(1) dissolving chloroauric acid trihydrate and aminophenol in water, wherein the molar concentration of the chloroauric acid trihydrate is 1-10mM, the molar concentration of the aminophenol is 1-10mM, adding triethylamine and tween 80, the final concentration of the triethylamine is 0.1-0.15%, the final concentration of the tween 80 is 0.5-1g/L, and mixing for 5-15min at the temperature of-5-5 ℃;

(2) dissolving 2-5g/L sodium borohydride in water, dropwise adding the sodium borohydride into the reaction solution in the step (1) under the stirring of 800-1200r/min of rotation speed, reacting for 1.5-3h, dialyzing for 20-30h by using a dialysis bag, wherein the molecular weight cut-off of the dialysis bag is 12-15kDa, and filtering and sterilizing by using a 0.22 micrometer filter to obtain the antibacterial nano material.

10. Use of an antibacterial nanomaterial according to claim 1 or 2 in the preparation of an antibacterial composition or an antibacterial medicament, preferably in the preparation of a medicament for the treatment of bacterial infections with aminoglycoside antibiotics.

Technical Field

The invention belongs to the field of biological materials, particularly relates to an antibacterial nano material and a preparation method and application thereof, and particularly relates to an aminophenol modified gold nanoparticle with an antibacterial effect and a preparation method and application thereof.

Background

The increase of antibiotic resistance is a major public health problem, and the prevalence of multidrug resistant strains has become a serious problem, preventing the treatment of serious infectious diseases worldwide. It is known that more than 70% of bacteria are resistant to one or more antibiotics. Gram-negative bacteria (e.g., E.coli) can cause urinary tract, gastrointestinal tract, and lung infections, while gram-positive bacteria (e.g., Staphylococcus aureus) can cause infections of skin and cartilage tissue.

Aminoglycoside antibiotics are a broad spectrum antibiotic that has been widely used since 1944 to treat various bacterial infections and life-threatening infectious diseases. Like other antibiotics, misuse and abuse can lead to the development of resistance by important microbial pathogens. In addition, aminoglycosides are positively charged at physiological pH, which leads to increased biotoxicity, such as nephrotoxicity, ototoxicity and neuromuscular blockade. These disadvantages have greatly hindered the clinical use of aminoglycoside drugs. Aminophenol (aminofenol) is a pharmaceutical intermediate for the production of antitubercular drugs, with a similar structure to the key unit of aminoglycoside antibiotics.

CN101225093A discloses a novel aminoglycoside derivative which has a broad antibacterial spectrum and high antibacterial activity compared with the existing aminoglycoside antibiotics, particularly has high antibacterial activity on drug-resistant bacteria and has good clinical use value. The prepared aminoglycoside compound and salt thereof are used as the medicine composition of necessary active ingredients for treating and/or preventing infectious diseases, but the method is complex to prepare, and the dosage for treating the infection in mice is up to 60 mg/kg.

Due to the increase of clinical and market demands, the development of novel antibacterial agents is imminent, and scholars at home and abroad prepare various antibacterial agents from various materials in the aspect of bacterial infection. The nano material has the characteristics of large specific surface area, high surface functionalization degree and unique physical and chemical properties, and provides great possibility for developing novel antibacterial drugs. Among them, gold nanoparticles have the advantages of low toxicity, easy functionalization, multivalence, easy detection, photothermal activity and the like, and are considered as one of the best antibacterial candidate materials.

CN108210515A discloses a novel nano-gold composite antibacterial agent and a preparation method thereof. The novel nano-gold composite antibacterial agent is nano-gold particles loaded with gentamicin sulfate. The method comprises the steps of adding polyvinylpyrrolidone into chloroauric acid, adding gentamicin sulfate after uniformly stirring, then adding a sodium borohydride solution to reduce the chloroauric acid solution, loading gentamicin sulfate, and removing the unloaded gentamicin sulfate in the solution through dialysis to obtain the nano-gold composite antibacterial agent. The obtained nano-gold composite antibacterial agent has good antibacterial effect on gram-positive bacteria, gram-negative bacteria and fungi. However, the use of antibiotics cannot be reduced fundamentally, the generation of bacterial drug resistance cannot be avoided, and the nano gold particles essentially only play a role of a drug carrier, so that the dispersion and the flow of gentamicin sulfate in the body are facilitated.

CN103242525A discloses a poly-o-hydroxyphenol antibacterial material and a preparation method thereof, wherein o-aminophenol is used as a raw material, and the poly-o-aminophenol macromolecular antibacterial material is synthesized by chemical oxidation in an acid solution. However, the obtained poly-o-aminophenol antibacterial material is in a micron-sized size and is not easy to circulate and metabolize in the body, and the preparation process thereof requires the use of organic solvents such as strong acids, which may cause biotoxicity and environmental pollution if remaining in the body.

Aiming at the defects that the existing antibacterial material is easy to generate drug resistance, poor in biological safety, undefined in antibacterial target and mechanism, complex in preparation method, large in size, not beneficial to in vivo circulation, difficult to store and the like, the field needs to develop a nano-grade antibacterial nano-material with excellent biocompatibility and a definite antibacterial mechanism to solve the infection problem generated by multi-drug resistant bacteria.

Disclosure of Invention

In view of the problems in the prior art, the invention provides an antibacterial nano material and a preparation method and application thereof, the antibacterial nano material is simple and convenient in preparation method, has better biological safety, and can effectively aim at multi-drug resistant bacterial infection.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an antibacterial nanomaterial, wherein the antibacterial nanomaterial is gold nanoparticles modified by aminophenol, and the aminophenol is modified on the surfaces of the gold nanoparticles through gold-ammonia bonds.

In the invention, aminophenol containing amino and hydroxyl functional groups is selected to be combined with the gold nanoparticles, and the aminophenol has no antibacterial activity and can play an antibacterial role after being combined with the gold nanoparticles. The multiple hydroxyl groups on the surface of the obtained material can target polysaccharides on the surface of bacteria, the permeability of cell membranes is increased by destroying the cell walls of the bacteria, the antibacterial target is definite, the aminophenol is used as a functional group to be combined with 16S ribosomal RNA of the bacteria, the synthesis of protein is inhibited, the bacteria are further killed, a better antibacterial effect is achieved, and particularly the antibacterial effect on aminoglycoside antibiotic resistant bacteria is better; meanwhile, the surface of the antibacterial nano material is negatively charged, and the antibacterial nano material has excellent biocompatibility.

As a preferred embodiment of the present invention, the aminophenol includes any one or a combination of at least two of o-aminophenol (2AP), p-aminophenol (4AP) or m-aminophenol (3 AP). Ortho-aminophenol, meta-aminophenol and para-aminophenol may all be combined with the gold nanoparticles to form the biomimetic antibiotics provided by the present invention.

Meanwhile, when any two or three of them are mixed and combined on the surface of the gold nanoparticle, the theoretically obtained gold nanoparticle also has an antibacterial effect, but whether or how the antibacterial effect changes cannot be predicted, and because the combination ratio of various aminophenols and the influence of some microscopic elements cannot be predicted, the change caused by which reason can not be determined even if the experimental result is obtained, therefore, in the invention, it is preferable to combine a single aminophenol on the surface of the gold nanoparticle.

Preferably, the gold nanoparticles are prepared by a sodium borohydride reduction method. The gold nanoparticles are prepared by a sodium borohydride reduction method during preparation, aminophenol can be modified to the surfaces of the gold nanoparticles while the gold nanoparticles are formed, the forming process and the modification process can be carried out in one step, so that the preparation method is simple, the modification efficiency is high, and the finally obtained particles have good antibacterial effect.

Preferably, the particle size of the antibacterial nano material is 2-8 nm, such as 2nm, 3nm, 4nm, 5nm, 6nm, 7nm or 8 nm. The obtained antibacterial nano material has small particle size, is nano-scale, and is easy to circulate and metabolize in vivo.

In a second aspect, the present invention provides a method for preparing the antibacterial nanomaterial, comprising the following steps: and mixing aminophenol with a reaction solution for preparing the gold nanoparticles, and reacting to obtain the antibacterial nano material.

The preparation method provided by the invention is simple, a sodium borohydride reduction method is specifically selected from various gold nanoparticle preparation methods, the antibacterial nanomaterial is prepared by a one-step method, namely, the gold nanoparticle is prepared while the antibacterial nanomaterial is modified by aminophenol, the preparation method is simple and quick, the combination of the aminophenol and the gold nanoparticle is facilitated, the modification degree is high, and the antibacterial effect of the obtained nanomaterial is also good.

As a preferred technical scheme of the invention, the preparation method comprises the following steps:

(1) mixing chloroauric acid, aminophenol, a pH regulator, a stabilizer and water, and dissolving to obtain a reaction solution;

(2) and (2) adding sodium borohydride into the reaction solution obtained in the step (1) to react, dialyzing, and sterilizing to obtain the antibacterial nano material.

As a preferred embodiment of the present invention, the molar concentration of chloroauric acid in step (1) is 1 to 10mM, and may be, for example, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, or 9 mM.

Preferably, the molar concentration of the aminophenol is 1 to 10mM, and may be, for example, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, or 9mM, or the like.

Preferably, the pH adjusting agent of step (1) comprises triethylamine. In the invention, the pH regulator is used for regulating the solution to be alkalescent so as to enhance the solubility of the aminophenol, and other alkalescent materials can be used for substitution.

Preferably, the final concentration of triethylamine is 0.1-0.15%, and may be, for example, 0.105%, 0.11%, 0.115%, 0.12%, 0.125%, 0.13%, 0.135%, 0.14%, 0.145%, or the like.

Preferably, the stabilizing agent of step (1) comprises tween 80. Tween 80 is a surfactant, and can stabilize the synthesized gold nanoparticles for a long time and facilitate long-term storage.

Preferably, the final concentration of Tween 80 is 0.5-1g/L, and may be, for example, 0.55g/L, 0.6g/L, 0.65g/L, 0.7g/L, 0.75g/L, 0.8g/L, 0.85g/L, 0.9g/L, 0.95g/L, or the like.

Preferably, the temperature for dissolving in step (1) is-5 to 5 ℃, for example, -4 ℃, -3 ℃, -2 ℃, -1 ℃, 0 ℃, 1 ℃, 2 ℃, 3 ℃ or 4 ℃, preferably 0 ℃, i.e. the dissolving in step (1) can be performed in an ice-water bath. Illustratively, the specific operation of step (1) is as follows: dissolving chloroauric acid trihydrate and aminophenol in water, wherein the molar concentration of the chloroauric acid trihydrate is 1-10mM, the molar concentration of the aminophenol is 1-10mM, adding triethylamine and tween 80, the final concentration of the triethylamine is 0.1-0.15%, and the final concentration of the tween 80 is 0.5-1g/L, and mixing for 5-15min under the condition of ice-water bath.

As a preferable technical scheme of the invention, the sodium borohydride in the step (2) is dissolved in water and then added into the reaction liquid in the step (1) under the condition of stirring.

Preferably, the concentration of the sodium borohydride dissolved in water is 2-5g/L, such as 2.2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L or 4.8 g/L.

Preferably, the rotation speed during stirring is 800-1200r/min, such as 820r/min, 850r/min, 900r/min, 950r/min, 1000r/min, 1050r/min, 1100r/min, 1150r/min or 1180 r/min.

Preferably, the reaction time in step (2) is 1.5-3h, such as 1.6h, 1.8h, 2h, 2.2h, 2.4h, 2.5h, 2.8h or 2.9 h.

Preferably, the reaction temperature in step (2) is-5 to 5 ℃, for example, -4 ℃, -3 ℃, -2 ℃, -1 ℃, 0 ℃, 1 ℃, 2 ℃, 3 ℃ or 4 ℃, preferably 0 ℃, i.e. the reaction in step (2) can be carried out in an ice-water bath.

As a preferable embodiment of the present invention, in the step (2), dialysis is performed using a dialysis bag.

Preferably, the cut-off molecular weight of the dialysis bag is 12-15kDa, and may be, for example, 12.2kDa, 12.5kDa, 12.8kDa, 13kDa, 13.5kDa, 13.8kDa, 14kDa, 14.5kDa, 14.8kDa or the like.

Preferably, the dialysis time in step (2) is 20-30h, such as 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h or 29 h.

Preferably, the sterilization method in step (2) is filtration sterilization using a 0.22 μm filter.

As a preferred technical scheme of the invention, the preparation method comprises the following steps:

(1) dissolving chloroauric acid trihydrate and aminophenol in water, wherein the molar concentration of the chloroauric acid trihydrate is 1-10mM, the molar concentration of the aminophenol is 1-10mM, adding triethylamine and tween 80, the final concentration of the triethylamine is 0.1-0.15%, the final concentration of the tween 80 is 0.5-1g/L, and mixing for 5-15min at the temperature of-5-5 ℃;

(2) dissolving 2-5g/L sodium borohydride in water, dropwise adding the sodium borohydride into the reaction solution in the step (1) under the stirring of 800-1200r/min of rotation speed, reacting for 1.5-3h, dialyzing for 20-30h by using a dialysis bag, wherein the molecular weight cut-off of the dialysis bag is 12-15kDa, and filtering and sterilizing by using a 0.22 micrometer filter to obtain the antibacterial nano material.

In a third aspect, the use of the antibacterial nanomaterial of the first aspect in the preparation of an antibacterial composition or an antibacterial medicament, preferably in the preparation of a medicament for the treatment of bacterial infection resistant to aminoglycoside antibiotics.

The antibacterial nano material is suitable for various infection, preventive treatment or public place disinfection and the like, is expected to be applied to the preparation of medical instruments, safe cosmetics and burn dressings, can be used in the aspects of water treatment or food preservation and the like, and can be used for preparing antibacterial drugs for treating abdominal infection.

The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.

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

(1) the antibacterial nano material AP _ Au NPs provided by the invention is an aminophenol modified gold nanoparticle, wherein the aminophenol is a drug intermediate; the AP _ Au NPs are used as a bionic antibiotic, are small in size and nano-scale, do not use an organic solvent system, have negative charges on the surface, have excellent biocompatibility and do not induce bacteria to generate drug resistance; compared with nephrotoxic drugs such as aminoglycoside drugs (such as gentamicin), the compound has higher biological safety;

(2) the antibacterial nano material provided by the invention has an accurate antibacterial mechanism, has a definite antibacterial target, can be combined with 16S ribosomal RNA to block the synthesis of bacterial protein, has a broad-spectrum antibacterial effect, has an excellent effect on aminoglycoside antibiotic-resistant bacteria, and can be used for treating various aminoglycoside antibiotic-resistant bacteria; the antibacterial nano material can also destroy bacterial cell walls which can not be destroyed by gentamicin, and has obvious effect on multidrug resistant bacteria;

(3) the preparation method of the aminophenol modified gold nanoparticle provided by the invention is simple, and the synthesized particle is stable and easy to store, and has the potential of large-scale production and wide clinical application prospect.

Drawings

FIG. 1 is a schematic diagram of the antibacterial principle of the aminophenol modified gold nanoparticles designed and synthesized in the present invention.

Fig. 2(a) is a topography map (50 nm scale) of the antibacterial nanomaterial prepared in example 1.

Fig. 2(b) is a topography map (scale 20nm) of the antibacterial nanomaterial prepared in example 1.

Fig. 3(a) is a topography map (50 nm scale) of the antibacterial nanomaterial prepared in example 2.

Fig. 3(b) is a topography map (20 nm scale) of the antibacterial nanomaterial prepared in example 2.

Fig. 4(a) is a topography map (50 nm scale) of the antibacterial nanomaterial prepared in example 3.

Fig. 4(b) is a topography profile (20 nm scale) of the antibacterial nanomaterial prepared in example 3.

Fig. 5 is a graph of uv-vis absorption spectra of the antibacterial nanomaterials provided in examples 1-3.

FIG. 6(a) is a histogram of the absorbance at 560nm of different concentrations of 2AP _ Au NPs.

FIG. 6(b) is a histogram of the absorbance at 560nm of different concentrations of 3AP _ Au NPs.

FIG. 6(c) is a histogram of absorbance at 560nm for different concentrations of 4AP _ Au NPs.

FIG. 7(a) is a scanning electron microscope characterization chart (scale 1 μm) obtained after S.a and MRSA were treated as a sample in test example 1.

Fig. 7(b) is a scanning electron microscope characterization chart (scale 1 μm) obtained after sample treatment of e.coli and MDR e.coli in test example 1.

FIG. 7(c) is a transmission electron microscope characterization chart (scale 0.2 μm) obtained after sample treatment S.a and MRSA and dehydration in test example 1.

Fig. 7(d) is a transmission electron microscope characterization chart (scale 0.5 μm) obtained after sample treatment e.coli and MDR e.coli in test example 1 and dehydration.

FIG. 8 is a photograph showing the result of detection by gel electrophoresis in test example 2.

FIG. 9(a) is a graph showing the survival rate of mice at various time points under S.a infection in test example 3.

FIG. 9(b) is a graph showing the survival rate of mice at different time points under MRSA infection in test example 3.

Fig. 9(c) is a graph showing survival rates of mice at different time points under e.coli infection in experimental example 3.

Fig. 9(d) is a graph showing survival rates of mice at different time points under MDR e.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

In the following examples, the chloroauric acid trihydrate has a molecular weight of 393.83, available from national pharmaceutical group chemical agents, ltd; scanning electron microscope (SEM, SU8220, HITACHI, Japan); transmission electron microscopy (TEM, Tecnai G220S-TWIN, FEI company, USA); dialysis bags (14kDa MW cut-off, Solarbio);

the schematic diagram of the design of the invention is shown in figure 1: firstly, preparing an antibacterial nano material, modifying aminophenol on the surface of a gold nanoparticle through a gold-ammonia bond, enabling one gold nanoparticle to be combined with a plurality of aminophenols as functional groups, injecting the obtained gold nanoparticle into a mouse body infected by bacteria, enabling the gold nanoparticle to target polysaccharide on the surface of the bacteria through polyhydroxy on the surface, destroying the cell wall of the bacteria, increasing the permeability of a cell membrane, combining the aminophenol with 16S rRNA in a ribosome 30S subunit, inhibiting protein synthesis and further killing the bacteria.

In the following examples, three antimicrobial nanomaterials are provided, including ortho-, meta-, and para-aminophenol modified gold nanoparticles, and are denoted as 2AP _ Au NPs, 3AP _ Au NPs, and 4AP _ Au NPs, respectively.