WO2023070886A1 - 一种抗细菌生物膜感染的Cu2WS4纳米颗粒及其光催化特性在细菌生物膜感染中的用途 - Google Patents

一种抗细菌生物膜感染的Cu2WS4纳米颗粒及其光催化特性在细菌生物膜感染中的用途 Download PDF

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WO2023070886A1
WO2023070886A1 PCT/CN2021/138543 CN2021138543W WO2023070886A1 WO 2023070886 A1 WO2023070886 A1 WO 2023070886A1 CN 2021138543 W CN2021138543 W CN 2021138543W WO 2023070886 A1 WO2023070886 A1 WO 2023070886A1
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biofilm infection
bacterial biofilm
nanoparticles
bacterial
infection
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张玉倩
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中国科学院深圳先进技术研究院
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  • the invention belongs to the intersecting field of nanotechnology and biomedicine, and in particular relates to the application of photocatalytic properties of nanoparticles in the treatment of bacterial biofilm infection.
  • Bacterial drug resistance mainly comes from two aspects: the acquired drug resistance at the bacterial cell level and the inherent environmental tolerance of bacterial biofilms.
  • the research and development of traditional antibiotics is carried out by screening synthetic compounds and natural compounds that can inhibit free bacteria.
  • the types of known compounds are limited, and the synthesis of natural compounds is difficult, which leads to difficulties in the development of antibiotics; on the other hand, targeting free bacteria
  • Antibiotics have very limited effects on bacterial biofilms, and cannot avoid the generation of drug resistance at the bacterial cell level. Therefore, new antibacterial agents that can replace antibiotics and are less likely to induce drug resistance have become the focus of research.
  • Nanomaterials exhibit excellent antibacterial properties. Nanomaterials can destroy the integrity of bacteria through direct contact with them, generate active oxygen, and kill bacteria through photothermal and photodynamic pathways. They are completely different from the mechanism of action of traditional antibiotics that interfere with bacterial metabolism. induce drug resistance.
  • nano-antibacterial agents the application of direct contact nano-antibacterial agents in complex physiological environments is limited; the application of photothermal and photodynamic nano-antibacterial agents that require near-infrared or ultraviolet light sources requires light source equipment, which is costly and expensive. Application is limited.
  • the problem to be solved by the present invention is how to realize the contact antibacterial effect under ordinary visible light irradiation under simpler and general conditions.
  • the Cu 2 WS 4 nanoparticles with excellent photocatalytic activity under visible light irradiation provided by the present invention have great advantages in this aspect. Under visible light irradiation, Cu 2 WS 4 nanoparticles can efficiently generate active oxygen and kill bacteria , Inhibit the formation of bacterial biofilm and promote wound healing.
  • One aspect of the present invention provides a Cu 2 WS 4 nanoparticle resistant to bacterial biofilm infection, the crystals are prepared by the following method:
  • the heating reaction temperature in S3) is 120-160°C.
  • the concentration of the (NH 4 ) 2 WS 4 aqueous solution is 1-100 mM/L, preferably 10 mM/L.
  • the concentration of the CuBr solution was dispersed in 9 mL of 0.1 M thioglycolic acid aqueous solution per 1 mM CuBr.
  • Another aspect of the present invention provides the use of the above-mentioned Cu 2 WS 4 nanoparticles for resisting bacterial biofilm infection in the preparation of a drug for treating bacterial biofilm infection.
  • the bacterial biofilm infection is Staphylococcus aureus biofilm infection.
  • Another aspect of the present invention provides the use of the above-mentioned Cu 2 WS 4 nanoparticles for anti-bacterial biofilm infection in the preparation of medicines for promoting wound healing.
  • Another aspect of the present invention provides the use of the above-mentioned Cu 2 WS 4 nanoparticles for anti-bacterial biofilm infection in the preparation of drugs for inhibiting bacterial drug-resistant bacteria.
  • the drug-resistant bacteria are Staphylococcus aureus drug-resistant bacteria.
  • Yet another aspect of the present invention provides a drug for promoting wound healing, said drug comprising the above-mentioned Cu 2 WS 4 nanoparticles for anti-bacterial biofilm infection as an active ingredient.
  • Still another aspect of the present invention provides a method for promoting wound healing, the method comprising applying the above-mentioned Cu 2 WS 4 nanoparticles for anti-bacterial biofilm infection on the surface of the wound.
  • Another aspect of the present invention provides a method for inhibiting bacterial biofilm infection, the method comprising applying the above-mentioned anti-bacterial biofilm infection Cu 2 WS 4 nanoparticles on the surface of bacterial biofilm infection.
  • the invention discloses a method for treating bacterial biofilm infection based on the photocatalytic properties of Cu 2 WS 4 nanoparticles. As shown in Fig. 1, under visible light irradiation, Cu2WS4 nanoparticles with a bandgap of 2.45 eV can efficiently generate reactive oxygen species, kill bacteria, inhibit bacterial biofilm formation, treat bacterial biofilm infection at wounds and promote wound healing. heal.
  • the invention is based on the excellent photocatalytic properties of Cu 2 WS 4 nanoparticles under visible light to kill Staphylococcus aureus and inhibit its biofilm formation, and use Cu 2 WS 4 nanoparticles to treat wound Staphylococcus aureus biofilm infection and promote wound healing .
  • Figure 1 Schematic diagram of Cu 2 WS 4 nanoparticles producing active oxygen to kill bacteria and inhibit bacterial biofilm formation under visible light irradiation.
  • Fig. 2 Schematic diagram of Cu 2 WS 4 nanoparticle structure.
  • FIG. 1 Characterization of basic properties of Cu 2 WS 4 nanoparticles.
  • Figure 4 Inhibition of S. aureus biofilm formation by Cu2WS4 nanoparticles.
  • (d) Image and (e) activity curve of S. aureus biofilm are examples of S. aureus biofilm.
  • FIG. 1 Treatment of S. aureus biofilm infection by Cu2WS4 nanoparticles.
  • FIG. 1 is a schematic diagram of the structure of Cu 2 WS 4 nanoparticles.
  • a is the TEM image of Cu 2 WS 4 nanoparticles
  • b is the high-resolution TEM image of Cu 2 WS 4 nanoparticles
  • c is the high-angle dark field scanning TEM image and elemental mapping image of Cu 2 WS 4 nanoparticles.
  • Staphylococcus aureus was recovered and stored in LB medium plate for a short period of time. Before use, a single colony was picked and inoculated in LB medium, and cultured with shaking at 37°C and 220 rpm for 12 hours; Staphylococcus aureus cultured overnight was supplemented with 1% Glucose LB medium was adjusted to a concentration of 2 ⁇ 10 7 CFU/mL, and added to a 96-well plate, 100 ⁇ L per well; 100 ⁇ L of different concentrations of Cu 2 WS 4 nanoparticle dispersion (added with 1 LB culture medium dilution of % glucose);
  • Example 3 Therapeutic Effect of Cu 2 WS 4 Nanoparticles on Staphylococcus aureus Biofilm Infected Wound Animal Test
  • mice were randomly divided into two groups, given physiological saline gel and Cu 2 WS 4 nanoparticle gel respectively;
  • a is a picture of a Staphylococcus aureus biofilm-infected wound
  • b is the area statistics
  • c is the bacterial colony forming unit of the infected site after 4 days of treatment
  • d is a microscopic image of the pathological section of the infected site after 4 days of treatment.
  • the present invention is based on the excellent photocatalytic properties of Cu 2 WS 4 nanoparticles under visible light to kill Staphylococcus aureus and inhibit its biofilm formation, and uses Cu 2 WS 4 nanoparticle gel to treat Staphylococcus aureus biofilm infection in wounds and Promotes wound healing.

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Abstract

一种抗细菌生物膜感染的Cu 2WS 4纳米颗粒及其光催化特性在细菌生物膜感染中的用途。一种抗细菌生物膜感染的Cu 2WS 4纳米颗粒及其制备方法，所述的抗细菌生物膜感染Cu 2WS 4纳米颗粒在制备治疗细菌生物膜感染、促进伤口愈合、抑制细菌耐药菌的药物中用途。上述纳米颗粒仅需可见光即可释放大量活性氧实现抑菌效果，能够促进伤口快速愈合。

Description

一种抗细菌生物膜感染的Cu2WS4纳米颗粒及其光催化特性在细菌生物膜感染中的用途 技术领域
本发明属于纳米技术和生物医药的交叉领域，特别是涉及一种纳米颗粒的光催化特性在细菌生物膜感染的治疗中的应用。
 背景技术
随着抗生素的广泛应用，细菌产生的耐药性已成为临床上的难题之一，给人类的健康带来了巨大的威胁。细菌的耐药性主要来源于两个方面：细菌细胞水平的获得性耐药性和细菌生物膜固有的环境耐受性。传统抗生素的研发是通过筛选可抑制游离态细菌的合成化合物和天然化合物进行，一方面，已知化合物的种类有限，而天然化合物合成困难，导致抗生素的研发困难；另一方面，以游离细菌为目标的抗生素对细菌生物膜的作用非常有限，并且无法避免细菌细胞水平耐药性的产生。因此，可替代抗生素且不易诱发耐药性的新型抗菌剂成为研究重点。
多种纳米材料表现出优异的抗菌性能，纳米材料可通过与细菌直接接触破坏其完整性、产生活性氧、光热和光动力途径杀死细菌，与传统抗生素干扰细菌新陈代谢的作用机制截然不同，不易诱发耐药性。
当前开发的纳米抑菌剂中，直接接触类纳米抗菌剂在复杂的生理环境中应用受限；需要近红外或紫外光源的光热和光动力类纳米抗菌剂的应用需光源仪器，成本较高且应用受限。
 技术问题
针对现有技术的方案，本发明所要解决的问题是如何在更简单通用的条件下，实现普通可见光照射下实现接触性抗菌效果。本发明提供的在可见光照射下具有优异光催化活性的Cu 2WS 4纳米颗粒在这一方面表现出极大的优势，在可见光照射下，Cu 2WS 4纳米颗粒高效产生活性氧，杀死细菌，抑制细菌生物膜的形成，促进伤口愈合。
技术解决方案
本发明一个方面提供了一种抗细菌生物膜感染的Cu 2WS 4纳米颗粒，所述晶体通过以下方法制备：
S1)配制CuBr溶液：将CuBr分散在巯基乙酸水溶液中；
S2) 配制 (NH 4) 2WS 4溶液：在(NH 4) 2WS 4的水溶液中加入NH 3·H 2O，获得(NH 4) 2WS 4溶液：
S3)将S1）和S2)获得的溶液混合后加热反应，分离后获得Cu 2WS 4纳米颗粒。
进一步地，S3)中加热反应温度为120-160℃。
进一步地，(NH 4) 2WS 4的水溶液的浓度为1-100 mM/L，优选为10mM/L。
进一步地，CuBr溶液的浓度为每1mMCuBr分散于9mL 0.1 M巯基乙酸水溶液中。
本发明另一个方面提供了上述抗细菌生物膜感染的Cu 2WS 4纳米颗粒在制备治疗细菌生物膜感染的药物中用途。
进一步地，所述细菌生物膜感染为金黄色葡萄球菌生物膜感染。
本发明另一个方面提供了上述抗细菌生物膜感染的Cu 2WS 4纳米颗粒在制备促进伤口愈合的药物中用途。
本发明另一个方面提供了上述抗细菌生物膜感染的Cu 2WS 4纳米颗粒在制备抑制细菌耐药菌的药物中用途。
进一步地，所述菌耐药菌为金黄色葡萄球菌耐药菌。
本发明再一个方面提供了一种促进伤口愈合的药物，所述药物包含上述抗细菌生物膜感染的Cu 2WS 4纳米颗粒作为活性成分。
本发明再一个方面提供了一种促进伤口愈合的方法，所述方法包括在伤口表面施用上述抗细菌生物膜感染的Cu 2WS 4纳米颗粒。
本发明再一个方面提供了一种抑制细菌生物膜感染的方法，所述方法包括在细菌生物膜感染表面施用上述抗细菌生物膜感染的Cu 2WS 4纳米颗粒。
本发明公开了一种基于Cu 2WS 4纳米颗粒光催化特性的细菌生物膜感染治疗方法。如图1所示，在可见光照射下，具有2.45 eV能带隙的Cu 2WS 4纳米颗粒可高效产生活性氧，杀死细菌，抑制细菌生物膜形成，治疗伤口处细菌生物膜感染并促进伤口愈合。
有益效果
发明基于Cu 2WS 4纳米颗粒在可见光下优异的光催化特性杀死金黄色葡萄球菌，抑制其生物膜形成，利用Cu 2WS 4纳米颗粒治疗伤口处金黄色葡萄球菌生物膜感染并促进伤口愈合。
附图说明
图1.在可见光照射下Cu 2WS 4纳米颗粒产生活性氧杀死细菌，抑制细菌生物膜形成示意图。
图2.Cu 2WS 4纳米颗粒结构示意图。（a）TEM图像；（b）高分辨TEM图像；（c）高角度环场暗场扫描TEM图像和元素映射图像。
图3.Cu 2WS 4纳米颗粒基本性质表征。（a）能带隙；（b）价带；（c）谱带位置和活性氧生成能力示意图；（d）活性氧诱导的对苯二甲酸荧光光谱。
图4. Cu 2WS 4纳米颗粒对金黄色葡萄球菌生物膜形成的抑制。（a）结晶紫染色的金黄色葡萄球菌生物膜照片；（b）结晶紫染色法定量测定金黄色葡萄球菌生物膜；（c）共聚焦显微镜拍摄金黄色葡萄球菌生物膜荧光图像；平板法计数金黄色葡萄球菌生物膜的（d）图像和（e）活性曲线。
图5. Cu 2WS 4纳米颗粒对金黄色葡萄球菌生物膜感染的治疗。金黄色葡萄球菌生物膜感染伤口的（a）图片和（b）面积统计；（c）治疗4天后感染部位细菌菌落形成单位；（d）治疗4天后感染部位病理切片的显微图像。
本发明的实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1Cu 2WS 4纳米颗粒的制备方法
将1mM (NH 4) 2WS 4溶解于100 mL超纯水中；将1mMCuBr分散于9mL 0.1 M巯基乙酸水溶液中；1将mLNH 3·H 2O加入搅拌中的(NH 4) 2WS 4溶液中，然后与CuBr溶液混合，分装入微波反应管中，140℃反应4小时；离心纯化获得Cu 2WS 4纳米颗粒。
对Cu 2WS 4纳米颗粒进行表征，结果见图2和图3；图2为Cu 2WS 4纳米颗粒结构示意图。其中a为Cu 2WS 4纳米颗粒TEM图像；b为Cu 2WS 4纳米颗粒高分辨TEM图像； c为Cu 2WS 4纳米颗粒高角度环场暗场扫描TEM图像和元素映射图像。
实施例2Cu 2WS 4纳米颗粒对金黄色葡萄球菌生物膜形成的细胞试验
金黄色葡萄球菌复苏和短期保存于LB培养基板，用前挑取单菌落接种于LB培养基，在37℃和220 rpm条件下振荡培养12小时；过夜培养的金黄色葡萄球菌用添加了1%葡萄糖的LB培养基调整浓度为2 × 10 7 CFU/mL，加入96孔板中，每孔100 μL；96孔板中分别加入100 μL不同浓度的Cu 2WS 4纳米颗粒分散液（添加了1%葡萄糖的LB培养基稀释）；
37℃静置孵育24小时；金黄色葡萄球菌生物膜形成的分析。结果见图4，其中a为结晶紫染色的金黄色葡萄球菌生物膜照片；b为结晶紫染色法定量测定金黄色葡萄球菌生物膜；c为共聚焦显微镜拍摄金黄色葡萄球菌生物膜荧光图像；d为平板法计数金黄色葡萄球菌生物膜的图像，e为活性曲线。
实施例3：Cu 2WS 4纳米颗粒对金黄色葡萄球菌生物膜感染伤口的治疗效果动物试验
6-8周龄的雌性Balb/c小鼠麻醉后，剃去背部毛发，建立一个直径约为4mm的伤口，并滴加100 μL浓度为1 × 10 7 CFU/mL的金黄色葡萄球菌；
小鼠随机分为两组，分别给予生理盐水凝胶和Cu 2WS 4纳米颗粒凝胶；
每日给药，4次后，将小鼠处死，分离伤口部位，计数金黄色葡萄球菌并利用病例切片查看愈合情况。结果见图5，其中a为金黄色葡萄球菌生物膜感染伤口的图片，b为面积统计；c为治疗4天后感染部位细菌菌落形成单位；d为治疗4天后感染部位病理切片的显微图像。
本发明基于Cu 2WS 4纳米颗粒在可见光下优异的光催化特性杀死金黄色葡萄球菌，抑制其生物膜形成，利用Cu 2WS 4纳米颗粒凝胶治疗伤口处金黄色葡萄球菌生物膜感染并促进伤口愈合。

Claims (9)

  1. 一种抗细菌生物膜感染的Cu 2WS 4纳米颗粒，其特征在于，所述晶体通过以下方法制备：
    S1)配制CuBr溶液：将CuBr分散在巯基乙酸水溶液中；
    S2) 配制 (NH 4) 2WS 4溶液：在(NH 4) 2WS 4的水溶液中加入NH 3·H 2O，获得(NH 4) 2WS 4溶液：
    S3)将S1）和S2)获得的溶液混合后加热反应，分离后获得Cu 2WS 4纳米颗粒。
  2. 权利要求1所述的抗细菌生物膜感染的Cu 2WS 4纳米颗粒在制备治疗细菌生物膜感染的药物中用途。
  3. 根据权利要求2所述用途，其特征在于，所述细菌生物膜感染为金黄色葡萄球菌生物膜感染。
  4. 权利要求1所述的抗细菌生物膜感染的Cu 2WS 4纳米颗粒在制备促进伤口愈合的药物中用途。
  5. 权利要求1所述的抗细菌生物膜感染的Cu 2WS 4纳米颗粒在制备抑制细菌耐药菌的药物中用途。
  6. 权利要求5所述用途，其特征在于，所述细菌耐药菌为金黄色葡萄球菌耐药菌。
  7. 一种促进伤口愈合的药物，其特征在于，所述药物包含权利要求1所述的抗细菌生物膜感染的Cu 2WS 4纳米颗粒作为活性成分。
  8. 根据权利要求7所述的药物，其特征在于，所述药物为外用药物。9、一种促进伤口愈合的方法，所述方法包括在伤口表面施用权利要求1所述的抗细菌生物膜感染的Cu 2WS 4纳米颗粒。
  9. 一种抑制细菌生物膜感染的方法，所述方法包括在细菌生物膜感染表面施用权利要求1所述的抗细菌生物膜感染的Cu 2WS 4纳米颗粒。
PCT/CN2021/138543 2021-10-25 2021-12-15 一种抗细菌生物膜感染的Cu2WS4纳米颗粒及其光催化特性在细菌生物膜感染中的用途 WO2023070886A1 (zh)

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