含银介孔二氧化硅-壳聚糖复合材料的制备与性能研究

doi:10.3877/cma.j.issn.1673-9450.2019.04.004

中华损伤与修复杂志(电子版) ›› 2019, Vol. 14 ›› Issue (04) : 256 -262. doi: 10.3877/cma.j.issn.1673-9450.2019.04.004

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论著

含银介孔二氧化硅-壳聚糖复合材料的制备与性能研究

夏德萌¹, 王胥人², 王元辰¹, 熊文韬³, 许硕贵¹, 周潘宇¹^,^†(

)

^1. 200433　上海，海军军医大学第一附属医院急诊科
^2. 200433　上海，海军军医大学护理学院
^3. 200433　上海，海军军医大学学员三大队

收稿日期:2019-07-02 出版日期:2019-08-01
通信作者: 周潘宇
基金资助:
国家自然科学基金(81571887); 上海市青年科技"启明星"(18QA1405400); 海军军医大学校级军事医学研究课题(2017JS15); 海军军医大学海洋军事医学重点攻关项目(2015HJ06)

Preparation and properties of silver-containing mesoporous silica-chitosan composites

Demeng Xia¹, Xuren Wang², Yuanchen Wang¹, Wentao Xiong³, Shuogui Xu¹, Panyu Zhou¹^,^†()

^1. Department of Emergency, First Affiliated Hospital of Naval Medical University, Shanghai 200433, China
^2. Department of Nursing, Naval Medical University, Shanghai 200433, China
^3. Naval Medical University Students Third Battalion, Shanghai 200433, China

Received:2019-07-02 Published:2019-08-01
Corresponding author: Panyu Zhou
About author:
Corresponding author: Zhou panyu, Email: panyu_zhou@163.com

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引用本文：

夏德萌, 王胥人, 王元辰, 熊文韬, 许硕贵, 周潘宇. 含银介孔二氧化硅-壳聚糖复合材料的制备与性能研究[J]. 中华损伤与修复杂志(电子版), 2019, 14(04): 256-262.

Demeng Xia, Xuren Wang, Yuanchen Wang, Wentao Xiong, Shuogui Xu, Panyu Zhou. Preparation and properties of silver-containing mesoporous silica-chitosan composites[J]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2019, 14(04): 256-262.

目的

探讨含银介孔二氧化硅-壳聚糖复合材料(Ag/MSN-Chi)的制备方法及其微观表征、细胞毒性、吸水性能、抗菌性能及止血性能。

方法

以正硅酸乙酯为前驱体，十六烷基三甲基溴化铵为致孔剂，采用离子交换法在介孔二氧化硅纳米粒子(MSN)中引入银离子，制备出具有抗菌作用的新型有序的含银介孔二氧化硅纳米粒子(Ag/MSN)材料。再利用烷基化壳聚糖负载Ag/MSN，制备出Ag/MSN-Chi。根据所用材料不同将实验分为实验组和空白对照组，实验组又分为3个亚组：MSN组、Ag/MSN组、Ag/MSN-Chi组，空白组为不加任何材料的阳性对照。计算MSN和Ag/MSN的比表面积、孔容、孔径和Ag/MSN与Ag/MSN-Chi的电荷。并通过吸水实验、体外凝血实验、抗菌实验对MSN、Ag/MSN和Ag/MSN-Chi的细胞毒性、吸水性能、止血性能及抗菌性能进行评价，计算细胞相对存活率、吸水率、凝血酶原时间(PT)、凝血活酶时间(APTT)及抑菌率。取健康成年新西兰大白兔18只，随机分成3组：对照组(采用医用纱布处理)、Ag/MSN组(采用Ag/MSN处理)、Ag/MSN-Chi组(采用Ag/MSN-Chi处理)，每组6只，建立肝创伤出血模型，计算止血时间。数据比较采用方差分析和t检验。

结果

MSN的比表面积为(523.8±12.4) m²/g、孔容为(1.2±0.4) m³/g、孔径为(3.5±0.9) nm；Ag/MSN的比表面积为(521.6±11.7) m²/g、孔容为(1.15±0.5) m³/g、孔径为(3.6±0.7) nm，2种材料的比表面积、孔容、孔径比较差异均无统计学意义(t＝0.224、0.135、0.015，P值均大于0.05)。经测量，Ag/MSN的Zeta电位为－19.7 mV，Ag/MSN-Chi的Zeta电位为10.27 mV，表明Ag/MSN表面电荷从负值变为正值。Ag/MSN-Chi组、Ag/MSN组和MSN组与小鼠成肌细胞共培养1、4、7 d的细胞相对存活率比较，差异均无统计学意义(F＝2.61、4.72、3.52, P值均大于0.05)。Ag/MSN组吸水率分别与MSN组和Ag/MSN-Chi组比较，差异均无统计学意义(t＝0.482、1.159，P值均大于0.05)。经检测，Ag/MSN-Chi组、Ag/MSN组、MSN组和空白对照组的PT比较，差异无统计学意义(F＝10.28，P>0.05)；Ag/MSN-Chi组、Ag/MSN组、MSN组和空白对照组APTT分别为(20.9±2.1)、(28.5±3.4)、(31.4±2.6)、(38.7±2.5) s，4组比较差异有统计学意义(F＝8.70，P<0.05)；Ag/MSN-Chi组、Ag/MSN组、MSN组APTT分别与空白对照组比较，差异均有统计学意义(t＝9.443、4.186、3.506，P值均小于0.05)；Ag/MSN-Chi组APTT与Ag/MSN组比较，差异有统计学意义(t＝3.294，P<0.05)。MSN组在培养0.5、2、4、6、24 h 5个时间点抑菌率比较差异无统计学意义(F＝5.437，P>0.05)；培养0.5 h，Ag/MSN组和Ag/MSN-Chi组抑菌率分别为(99.7±5.2)%、(97.1±5.4)%，与培养0.5 h MSN组抑菌率(11.2±5.8)%比较，差异均有统计学意义(t＝19.678、18.775, P值均小于0.05)；培养24 h，Ag/MSN组和Ag/MSN-Chi组抑菌率分别为(73.2±5.1)%和(72.9±6.9)%，与MSN组(11.8±5.7)%比较，差异均有统计学意义(t＝13.904、11.825, P值均小于0.05)。Ag/MSN-Chi组、Ag/MSN组和对照组止血时间分别为(12.3±1.5)、(17.2±3.4)、(28.1±3.8) s，3组比较差异有统计学意义(F＝5.892，P<0.05)；Ag/MSN-Chi组和Ag/MSN组止血时间分别与对照组比较，差异均有统计学意义(t＝9.473、5.236, P值均小于0.05)；且Ag/MSN-Chi组与Ag/MSN组止血时间比较，差异有统计学意义(t＝3.230，P<0.05)。

结论

Ag/MSN-Chi在不增加细胞毒性的基础上具有有较好的吸水性能、止血性能及抗菌性能。

关键词: 壳聚糖, 银, 止血, 介孔二氧化硅, 抗菌

Objective

To investigate the preparation method of silver-containing mesoporous silica nanoparticle-chitosan(Ag/MSN-Chi) composites and their microscopic representation, cytotoxicity, water absorption, rapid hemostatic properties and antibacterial properties.

Methods

Using ethyl orthosilicate as precursor and cetyltrimethylammonium bromide as porogen, silver ions were introduced into mesoporous silica nanoparticles (MSN) by ion-exchange method to prepare novel ordered silver-containing MSN (Ag/MSN) material with antibacterial action. Ag/MSN-Chi was prepared by using alkylated chitosan loaded with Ag/MSN. The experiment were divided into experimental group and blank control group according to the materials used. The experimental group was further divided into three subgroups: MSN group, Ag/MSN group and Ag/MSN-Chi group. The blank group was a positive control without material. The specific surface area, pore volume, pore size of MSN and Ag/MSN, the charge of Ag/MSN and Ag/MSN-Chi were calculated. The cytotoxicity, water absorption performance, hemostatic and antibacterial properties of MSN, Ag/MSN and Ag/MSN-Chi were evaluated by water absorption test, in vitro coagulation test and antibacterial test to calculate the relative cell survival rate, water absorption rate and prothrombin time (PT), activated partial thromboplastin time (APTT), and bacteriostatic rate. Eighteen healthy adult New Zealand white rabbits were randomly divided into three groups: control group (treated with medical gauze), Ag/MSN group (treated with Ag/MSN), and Ag/MSN-Chi group (treated with Ag/MSN-Chi), 6 rats in each group. A hepatic trauma hemorrhage model was established and the hemostasis time was calculated. Data comparisons were analyzed by analysis of variance and t test.

Results

The specific surface area of MSN was (523.8±12.4) m²/g, the pore volume was (1.2±0.4) m³/g, and the pore diameter was (3.5±0.9) nm; the specific surface area of Ag/MSN was (521.6±11.7) m²/g, the pore volume was (1.15±0.5) m³/g, and the pore diameter was (3.6±0.7) nm, there was no significant difference in specific surface area, pore volume and pore size between the two materials (t=0.224, 0.135, 0.015; with P values above 0.05). The Zeta potential of Ag/MSN was measured to be -19.7 mV, and the Zeta potential of Ag/MSN-Chi was 10.27 mV, indicating that the surface charge of Ag/MSN changed from a negative value to a positive value. The relative survival rates of Ag/MSN-Chi group, Ag/MSN group and MSN group at 1, 4, and 7 days after co-culture with mouse myoblasts were not statistically significant (F=2.61, 4.72, 3.52; with P values above 0.05). The water absorption of Ag/MSN group was significantly different from that of MSN group and Ag/MSN-Chi group (t=0.482, 1.159; with P values above 0.05). There was no significant difference in PT between Ag/MSN-Chi group, Ag/MSN group, MSN group and blank control group (F=10.28, P>0.05). The APTT of Ag/MSN-Chi group, Ag/MSN group, MSN group and blank control group were (20.9±2.1), (28.5±3.4), (31.4±2.6), (38.7±2.5) s, respectively, the difference of the 4 groups was statistically significant (F=8.70, P<0.05); the APTT of Ag/MSN-Chi group, Ag/MSN group and MSN group were significantly different from the blank control group, the differences were statistically significant (t=9.443, 4.186, 3.506; with P values below 0.05); the difference between Ag/MSN group and Ag/MSN-Chi group of APTT was statistically significant (t=3.294, P<0.05). There was no significant difference in the inhibition rate between the MSN group at 0.5, 2, 4, 6 and 24 h (F=5.437, P>0.05); after incubation for 0.5 h, the inhibition rates of Ag/MSN group and Ag/MSN-Chi group were (99.7±5.2)% and (97.1±5.4)%, respectively, compared with the incubation rate of 0.5 h in MSN group (11.2±5.8)%, the differences were statistically significant (t=19.678, 18.775; with P values below 0.05). After 24 h of culture, the inhibition rates of Ag/MSN group and Ag/MSN-Chi group were (73.2±5.1)% and (72.9±6.9)%, respectively, compared with MSN group (11.8±5.7)%, the differences were statistically significant significance (t=13.904, 11.825; with P values below 0.05). The hemostasis time of Ag/MSN-Chi group, Ag/MSN group and control group were (12.3±1.5), (17.2±3.4), (28.1±3.8) s, respectively, the difference between the three groups was statistically significant (F=5.892, P<0.05); the Ag/MSN-Chi group and the Ag/MSN group were significantly different from the control group (t=9.473, 5.236; with P values below 0.05); and the Ag/MSN-Chi group was compared with the Ag/MSN group, the difference was statistically significant (t=3.230, P<0.05).

Conclusion

Ag/MSN-Chi has better water absorption performance, hemostatic property and antibacterial property without increasing cytotoxicity.

Key words: Chitosan, Silver, Hemostasis, Mesoporous silica nanoparticle, Antibacterial

表1 Ag/MSN－Chi组、Ag/MSN组和MSN组小鼠成肌细胞相对存活率比较(%, ±s)

组别	样本数 (孔)	培养1 d	培养4 d	培养7 d
Ag/MSN－Chi组	3	109.0±4.3	107.0±5.2	101.0±4.9
Ag/MSN组	3	97.0±3.3	91.0±2.7	95.0±3.5
MSN组	3	103.0±3.1	109.0±4.3	102.0±4.7
F值	?	2.61	4.72	3.52
P值	?	>0.05	>0.05	>0.05

表1 Ag/MSN－Chi组、Ag/MSN组和MSN组小鼠成肌细胞相对存活率比较(%, ±s)

组别	样本数 (孔)	培养1 d	培养4 d	培养7 d
Ag/MSN－Chi组	3	109.0±4.3	107.0±5.2	101.0±4.9
Ag/MSN组	3	97.0±3.3	91.0±2.7	95.0±3.5
MSN组	3	103.0±3.1	109.0±4.3	102.0±4.7
F值	?	2.61	4.72	3.52
P值	?	>0.05	>0.05	>0.05

表2 Ag/MSN－Chi组、Ag/MSN组、MSN组和空白对照组APTT与PT比较(s, ±s)

组别	样本数(孔)	APTT	PT
Ag/MSN－Chi组	3	20.9±2.1^ab	12.3±2.1
Ag/MSN组	3	28.5±3.4^a	11.9±3.5
MSN组	3	31.4±2.6^a	12.4±2.7
空白对照组	3	38.7±2.5	12.3±3.4
F值	?	8.70	10.28
P值	?	<0.05	>0.05

表2 Ag/MSN－Chi组、Ag/MSN组、MSN组和空白对照组APTT与PT比较(s, ±s)

组别	样本数(孔)	APTT	PT
Ag/MSN－Chi组	3	20.9±2.1^ab	12.3±2.1
Ag/MSN组	3	28.5±3.4^a	11.9±3.5
MSN组	3	31.4±2.6^a	12.4±2.7
空白对照组	3	38.7±2.5	12.3±3.4
F值	?	8.70	10.28
P值	?	<0.05	>0.05

图1 Ag/MSN－Chi组、Ag/MSN组和MSN组不同培养时间对大肠杆菌的抑菌率变化。Ag/MSN－Chi为含银介孔二氧化硅纳米粒子－壳聚糖；Ag/MSN为含银介孔二氧化硅纳米粒子；MSN为介孔二氧化硅纳米粒子

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Preparation and properties of silver-containing mesoporous silica-chitosan composites

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Preparation and properties of silver-containing mesoporous silica-chitosan composites