生物合成纳米银/壳聚糖复合抑菌剂的制备与性能

吴承宗; 魏亚楠; 黄婷; 苗雨颖; 张译丹; 刘煜扬; 刘小莉; 赵彦翠; 乔洪金; 王磊

生物合成纳米银/壳聚糖复合抑菌剂的制备与性能

鲁东大学　生命科学学院, 烟台 264025

基金项目: 山东省自然科学基金项目(ZR2021 MC102)；烟台市科技发展计划(2022 XDRH010)

详细信息

通讯作者:
王磊，博士，教授，研究方向为纳米技术在病害防控中的应用　E-mail: wanglei9909@163.com

吴承宗和魏亚楠为共同第一作者，对本文具有同等贡献
中图分类号: TB333
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- 被引次数: 0
出版历程
- 收稿日期: 2022-11-14
- 修回日期: 2022-12-12
- 录用日期: 2023-01-08
- 网络出版日期: 2023-01-31

Biosynthesis and properties of AgNPs /chitosan composite antibacterial agent

College of Life Sciences, Ludong University, Yantai 264025, China

摘要

摘要: 纳米银(AgNPs)在水产养殖等抑菌领域中有着广阔的应用前景，但AgNPs粒径小、比表面积大容易产生团聚，且直接穿透正常的细胞膜，存在一定的细胞毒性。为解决上述问题本文以腊梅花瓣提取液生物合成纳米银，并与具有良好生物相容性的天然高分子材料壳聚糖(CS)络合制备出纳米银复合抑菌剂AgNPs/CS。制备出的产品能提高AgNPs的抑菌活性和稳定性，并具有理想的生物安全性，为AgNPs在水产养殖领域的应用开辟了新的途径。浓度为0.4 μg/mL的AgNPs/CS可使灿烂弧菌生物膜形成量减少74.5%；浓度为0.8 μg/mL的AgNPs/CS处理组可使生物膜形成量减少84.4%。因此，AgNPs/CS对灿烂弧菌生物膜具有显著的清除作用，为打破病原菌耐药性的瓶颈奠定基础。1AgNPs/CS 对灿烂弧菌生物膜的影响AgNPs/CS对293T细胞的毒性实验表明，在MIC和MBC分别为0.43 μg/mL和0.87 μg/mL的有效使用浓度下，细胞毒性评级为1级，具有较为理想的生物安全性。1AgNPs/CS浓度对体外细胞相对增值率和细胞毒性评级的影响

Concentration(μg/mL)	OD value	RGR(%)	cytotoxicity
0	0.896±0.126	100.00	0
0.25	0.847±0.085	94.534	1
0.5	0.832±0.073	92.818	1
1.0	0.768±0.094	85.707	1
5.0	0.639±0.082	71.317	2
10.0	0.568±0.065	63.393	2
20.0	0.492±0.071	54.911	2
40.0	0.061±0.041	6.885	4
80.0	0.046±0.023	5.154	4

关键词:

Abstract: In order to prepare a new type of safe, stable and efficient nano silver composite antibacterial agent, silver nanoparticles/chitosan (AgNPs/CS) was biosynthesized by by one-step method using Chimonanthus praeco petal extract and chitosan as reducing agent and stabilizing agent, respectively. The optimal preparation procedure was determined by single factor experiment. The products were characterized by UV-vis absorption spectroscopy, transmission electron microscopy and X-ray diffraction, and their antibacterial activity, anti-drug resistance, stability as well as biological safety were comprehensively evaluated. The experimental results showed that the AgNPs/CS had the characteristic absorption peak of AgNPs at 451 nm, and the evenly dispersed AgNPs were spherical with average diameter of 12.83 nm, and the crystals had a face-centered cubic structure. The AgNPs/CS show antibacterial activity against aquatic pathogens both in vitro and in vivo, and had excellent anti-drug resistance, biosafety and stability. Therefore, AgNPs/CS is an ideal composite antibacterial agent, which has a prospective application in aquaculture field.

Key words:

1) 吴承宗和魏亚楠为共同第一作者，对本文具有同等贡献

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图 1 纳米银反应条件的优化

Figure 1. Optimization of AgNPs reaction conditions

(a): Volume of chitosan ; (b): AgNO₃ concentration; (c): Volume of Chimonanthus praecox extract; (d): Reaction temperature; (e):Reaction time

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图 2 腊梅提取液、壳聚糖和纳米银/壳聚糖(AgNPs/CS)溶液的照片(a)及紫外吸收光谱图(b)

Figure 2. Photograph(a)and UV-vis absorption spectra (b) of Chimonanthus praecox extracts、chitosan and silver nanoparticles/chitosan (AgNPs/CS)

1:Chitosan; 2: Chimonanthus praecox extracts; 3:AgNPs/CS

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图 3 AgNPs/CS的透射电镜图(a)和粒径分布图(b)

Figure 3. TEM image(a)and particle size distribution (b)of AgNPs/CS

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图 4 AgNPs/CS的X射线衍射图谱

Figure 4. X-ray diffraction pattern of AgNPs/CS

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图 5 AgNPs/CS 对6种水产病原菌的抑菌圈实验

Figure 5. Inhibition zone test of AgNPs/CS against 6 aquatic pathogens

1: AgNPs/CS; 2: AgNPs; 3: Chimonanthus praecox extracts; 4: Amp; 5: Normal saline

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图 6 AgNPs/CS不同浓度下灿烂弧菌攻毒处理菲律宾蛤仔的死亡率

Figure 6. Fatality rate of R.philippinarum challenged with V.splendidus at different concentrations of AgNPs/CS

Note: "*" indicates significant difference; "**" indicates highly significant difference

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图 7 AgNPs/CS 对灿烂弧菌生物膜的影响

Figure 7. Effect of the biosynthesized AgNPs/CS on biofilm formation by V.splendidus (n=3)

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图 8 不同存放时间 AgNPs/CS和AgNPs紫外吸收光谱图

Figure 8. UV-Vis absorption spectra of AgNPs/CS ang AgNPs at different storage times

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图 9 AgNPs/CS和AgNP的Zeta 电位图

Figure 9. Zeta potential the biosynthesized AgNPs/CS and AgNPs

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1 AgNPs/CS浓度对体外细胞相对增值率和细胞毒性评级的影响

Concentration(μg/mL)	OD value	RGR(%)	cytotoxicity
0	0.896±0.126	100.00	0
0.25	0.847±0.085	94.534	1
0.5	0.832±0.073	92.818	1
1.0	0.768±0.094	85.707	1
5.0	0.639±0.082	71.317	2
10.0	0.568±0.065	63.393	2
20.0	0.492±0.071	54.911	2
40.0	0.061±0.041	6.885	4
80.0	0.046±0.023	5.154	4

下载: 导出CSV

表 1 对6种病原菌的抑菌圈直径(n=3)

Table 1. Diameter of inhibition zone against 6 pathogens(n=3)

Strain	Inhibition zone diameter
Strain	AgNPs/CS	AgNPs	Amp
A.punctata	19.24±0.47	16.73±0.53	16.25±0.86
V.alginolyticus	23.39±1.03	21.07±0.19	15.72±0.28
V.parahaemolyticus	24.52±0.76	23.35±1.08	23.17±0.10
V.anguillarum	22.01±0.37	19.93±0.11	15.35±0.28
V.harveyi	18.11±0.25	17.98±0.66	9.03±0.34
V.splendidus	26.59±0.65	22.93±2.26	21.2±1.47

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表 2 AgNPs对灿烂弧菌的最小抑菌浓度和最小杀菌浓度(n=3)

Table 2. MIC and MBC tests of AgNPs against V.splendidus(n=3)

Numbering	Concentration/(μg·mL⁻¹)	AgNPs/CS		Concentration/(μg·mL⁻¹)	AgNPs
Numbering	Concentration/(μg·mL⁻¹)	24 h	48 h	Concentration/(μg·mL⁻¹)	24 h	48 h
1	112.5	−	−	33.33	−	−
2	56.3	−	−	16.67	−	−
3	28.1	−	−	8.33	−	−
4	14.1	−	−	4.17	−	−
5	7.03	−	−	2.08	−	−
6	3.51	−	−	1.04	−	−
7	1.75	−	−	0.52	−	+
8	0.87	−	−	0.26	+	+
9	0.43	−	+	0.13	+	+
10	0.21	+	+	0.07	+	+

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表 3 AgNPs/CS对多次传代灿烂弧菌最小抑菌浓度的研究(n=3)

Table 3. MIC tests of AgNPs/CS against multigenerational V.splendidus

Passage number	MIC/(μg·mL⁻¹)
Passage number	AgNPs/CS	Amp
1	0.43	9.76
2	0.43	19.53
3	0.43	19.53
4	0.43	39.06
5	0.43	78.13
6	0.87	78.13
7	0.87	312.5
8	0.87	312.5
9	0.87	312.5
10	0.87	625
11	0.87	625
12	0.87	625
13	0.87	625
14	0.87	1250
15	0.87	1250
16	0.87	1250
17	0.87	1250
18	0.87	1250
19	0.87	1250
20	0.87	1250

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表 4 AgNPs/CS浓度对体外细胞相对增值率和细胞毒性评级的影响

Table 4. Effect of AgNPs/CS concentration on relative proliferation rate and cytotoxicity rating of cells in vitro

Concentration/(μg·mL⁻¹)	OD value	RGR(%)	cytotoxicity
0	0.896±0.126	100.00	0
0.25	0.847±0.085	94.534	1
0.5	0.832±0.073	92.818	1
1.0	0.768±0.094	85.707	1
5.0	0.639±0.082	71.317	2
10.0	0.568±0.065	63.393	2
20.0	0.492±0.071	54.911	2
40.0	0.061±0.041	6.885	4
80.0	0.046±0.023	5.154	4

下载: 导出CSV

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