中空载银纤维的制备及其抗菌和安全性能的研究
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摘要
几个世纪以来,金属银就被广泛地用于各种疾病的预防和治疗中,特别是在消炎、防感染方面。银离子具有极强的广谱抗菌抑菌作用,同时对人体细胞又是低毒副作用。银离子的抗菌作用主要是与带负电荷的细菌细胞壁的吸附作用,使细胞酶失活,破坏细胞膜透性,最终导致细菌凋亡或死亡。目前,新型载银抗菌聚合材料的研究成为了学术界和工业领域极为关注的课题之一。这种将热塑性材料与银的结合方式使得该材料同时具备了热塑性材料的优良加工性能和银固有的抗菌性能。而目前采用的抗菌载银材料的制备方法存在着或多或少的缺陷,例如耐洗性差、着色性等等。
本课题通过压力差作用和银镜反应制备得到具有抗菌性能的中空载银纤维——银纤维。初生态银粒子被置换出来而附着于纤维中空内表面层。通过SEM观察到银粒子的尺寸大约为0.1～0.5μm,且均匀地分布于纤维中空内表面层中。X-衍射分析观察到衍射峰只在20=38.2°,44.3°和64.5°处出现,这也证实了银粒子的存在以及银粒子的尺寸还没能大到使其衍射峰出现在2θ=77.4°和81.5°处。而这种尺寸的初生态银粒子具有极强的活性而使得该材料具有更强的抗菌性能。
银离子的释放是载银抗菌材料具有抗菌性的最基本条件。本课题制备的中空载银纤维中的银离子是通过纤维头尾两端释放。通过对银离子释放试验及其定性分析,研究了影响银纤维中银离子释放的因素,包括释放时间、纤维长度、银粒子含量以及纤维种类等。随着时间的增加,释放的银离子浓度逐渐增加并于3天后达到平衡;当纤维重量一定时,随着纤维的长度的增加,纤维根数减少,即能够释放银离子的头尾两端减少,因此银离子释放减少;银离子的释放量随着银粒子含量的增加而增加;不同种类的纤维其吸湿性不同,吸湿性较好的其银离子释放量也较多。
作为医用材料来说,银离子浓度是一个极为重要的参数。如果银离子浓度太高,就会对人体组织产生影响,如“银中毒”;如果太低,抗菌作用则受到限制。因此确定银离子浓度的阀值至关重要。本课题在Lotka-volterra模型的基础之上,建立银离子-细菌模型,确定了银离子浓度阈值,(?),其中,a值因菌种不同而不同,本文中金黄色葡萄球菌的a=0.82;λ值则与中空载银纤维的结构参数有关。这两个数据均可通过实验容易获得,从而计算得到银离子浓度阙值。而银纤维的抗菌性能试验表明银纤维具有广谱抗菌性及抗耐药菌性。影响抗菌效果的因素包括有:外界环境潮湿与否、纤维量及纤维结构参数等。当银纤维浓度为37.5μg/ml时,抗菌效果将达到90%以上。通过银纤维处理前后金黄色葡萄球菌和大肠埃希菌的SEM照片可以看出,银纤维处理后的细菌膨胀至崩裂,而且还发现银纤维对金黄色葡萄球菌的破坏作用大于大肠埃希菌,这可能是由于它们的细胞壁组成不同而造成的。
为了探讨中空载银纤维的细胞安全性,本课题选用了三种传代人系细胞株(Vero、Hela和WI-38)和一种原代细胞(从小儿包皮提取的上皮细胞)作为试验用人系细胞株进行安全性试验。试验结果表明,在银纤维浓度不超过4mg/ml时(包括抗菌浓度37.5μg/ml),银纤维的加入对人系细胞株的存活。而从电镜图片观察到,当加入银纤维达到4mg/ml时,细胞开始出现圆缩、凹陷等现象,这些现象可能是细胞死亡的一种前兆。
本课题还从免疫学和卫生毒理学的角度,通过皮肤覆盖和皮下埋植两种途径,检测了中空载银纤维对豚鼠的免疫水平(包括体液免疫、细胞免疫和非特异性免疫)和遗传物质的影响,并对主要器官,如心、肝、脾、肺、肾进行病理分析。通过对银纤维对豚鼠的免疫功能和遗传物质的影响的初步研究发现,当皮肤覆盖或埋植少于0.03g银纤维时,对免疫功能、遗传物质和主要器官都未造成损伤。但是当埋植高剂量银纤维,如0.03g时,溶血空斑细胞、循环免疫复合物和红细胞脆性都受到了影响,且病理切片分析也证实了埋植0.03g银纤维后,肝脏和肾脏开始出现病变。
综上所述,本课题通过压力差作用和银镜反应制备得到了抗菌中空载银纤维,并对银纤维的释放性能、抗菌性、细胞安全性及豚鼠毒理性进行了研究,并得出了相关结论,为该材料的实际应用提供了实验及理论基础。研究表明,该中空载银纤维具有很好的抗菌效果,当银纤维浓度达到37.5μm/ml时,抗菌效果就已经达到90%以上;当银纤维浓度在4mg/ml范围内时(包括抑菌浓度37.5μg/ml),银纤维的加入对人系细胞株的存活和形态均无影响;而当银纤维覆盖于豚鼠皮肤或埋植少于0.03g银纤维于豚鼠皮下时,银纤维的加入都不影响豚鼠的免疫功能、遗传物质及器官组织。但是当埋植超过0.03g银纤维后,造成了人系细胞株及豚鼠机体的轻微损伤。因此,我们在使用过程中应注意加强对载银材料的管理,尽快建立和完善载银材料安全性的研究方法和体系,重视载银材料的剂量-效应关系,为载银材料的应用提供可靠的依据。同时由于条件的限制,本课题未能将该材料制备成纺织产品,例如水刺无纺布,并进行性能研究,是本课题的不足之处,还有待于后续研究进一步完善。
Silver has been used for centuries to prevent and treat a variety of diseases, mostly notably infections. Previous work has shown that silver ions have strong inhibitory and bacterial effects as well as broad spectrum of activity, and concomitant low toxicity to mammalian cells. The inhibitory effect of silver ions was believed to be due to its sorption to the negatively charged bacterial cell wall, deactivating cellular enzymes, disrupting membrane permeability, and ultimately leading to cell lysis and death. Nowadays the introduction of new silver-based antibacterial polymers represented a great challenge for both academic world and industry. The silver-based thermoplastic polymer composites combined the excellent process properties of the thermoplastics with the inherent antibacterial ability of the silver. The products loaded with silver had disadvantages more or less, as lasting, staining, uncontrolled release, and et al.
Hollow fiber loaded with silver particles (silver fiber) was prepared in the study through the different press effect and the silver mirror reaction, and the silver was displaced and adhered on the fiber inner wall. The size of silver particles was approximately 0.1～0.5μm, which was measured by SEM test. X-ray of PET hollow fiber was compared to the silver fiber. The most important peaks are denoted clearly. The curve exhibited the obvious peaks at 2θ=38.2°, 44.3°, and 64.5°, which are due to the face-centered cubic crystalline silver, corresponding to the crystal faces of (111), (200), and (220) of silver, respectively. And the peaks at 2θ=77.4°and 81.5°, which are due to the crystal faces of (311) and (222) of silver, had not appeared. Therefore, it was very clear that the silver fiber was consisted of silver particles and the size of silver particles was small, which was consistent with SEM results.
When the silver fiber was entered in a water environment, the silver ions would be released form the two ends of the fiber. And the silver ions were the principle factor on the antibacterial efficiency. So the release of silver ions was investigated by release experiments and qualitative analysis. It was found that the concentration of silver ions released from the silver fiber was affected by many factors, such as release hours, fiber length, concentration of silver particles and fiber type. With the increase of release hours, the concentration of silver ions released from the silver fiber increased, and would be invariable after 3 days. And when there was the same weight of silver fiber, with the increase of the length of silver fiber, there were fewer points to release silver ions. And the silver ions released increased as well as the increase of concentration of silver particles. The study also indicated that the hollow fiber which had good moisture absorption released more concentration of silver ions.
In this paper, a silver ion-bacterium model was established to determine the threshold concentration of silver ions. The silver ion-bacterium model was similar to predator-prey model also known as the Lotka-Volterra model which described the dynamics of biology system in which two species interacted, one a predator and one its prey. Silver ions were antibacterial and treated as predators; bacteria were killed by silver ions and treated as preys. The concentration of silver ions was a critical parameter in medicine applications. When it was too high, it was lethal to the human organism, this was Argyria; while it was too low, the inhibitory efficiency was seriously affected. It was important to determine the threshold concentration of silver ions, , where a varied with different bacteria, andα=0.82 for S. aureus;λvaried with different hollow fibers and number of silver particles on the inner surface. They were easy to be got by experimental results.The antibacterial test showed that the silver fiber had great board-spectrum antibacterial activity, and had inhibitory effect on drug resistant bacteria. The antibacterial efficiency was dependent on the wateriness of the fibers, because the watery silver fiber released silver ions more than that of waterless. The antibacterial concentration of 38mm silver fiber was 37.5μg/ml. At that concentration, the inhibitory ratio reached up to 90%. From the SEM images of bacteria treated by silver fiber, it was found that the bacteria was expanded and cracked, and the different effect on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) may be due to the different configuration of cell walls of these two bacteria.
Four clay cell types were chosen to determine the cytotoxicity of the silver fiber, which were Vero, Hela, WI-38 and fibroblasts developed from primary culture of discarded surgical tissue, foreskins. And the experimental results showed that the cell proliferation was not affected by the concentration of silver fiber for antibacterial activity. And when treated by 4mg/ml silver fiber, the cell became less polyhedral, more fusiform, shrunken and rounded. It showed that cell proliferation and morphology of all cells was normal in the presence of the silver fiber down to a concentration of 4mg/ml, including 37.5μg/ml. However, abnormal size, shrinkage and rounded appearance of cells at higher dose (4mg/ml) suggested toxicity of the silver fiber.
During the process of the study, toxicity test on Guinea pig was conducted from the perspective of immunology and hygienic toxicology, in which two different contamination methods were used, including skin smearing and embedding. The effect of the silver fiber on immunity level, including humoral, cellular and non-special immunity, and germ plasm of Guinea pig were detected. Furthermore, pathological analysis was carried out on some primary organs, such as stomach, liver and kidney. Based on the elementary research on the effect of the silver fiber on immune function and germ plasm of the tested animals, it was found that in the presence of the silver fiber down to 0.03g, the silver fiber would not cause harm to the immune function, germ plasm and organs. But when in high dose, as 0.03g, hemolytie plaque-forming cell, immune complexes and red cell osmotic fragility was abnormal, and the pathological analysis further testified that the liver and kidney were really injured by 0.03g silver fiber. Therefore, the reasonable quantity control of the silver fiber during its application process is necessary, which requires that series of large-scale experiments need to be undertaken in further research process so as to acquire the dose-effect relation of silver fiber and provide reliable evidence for its extensive application.
The study prepared the hollow fiber loaded with silver particles and investigated its release property, antibacterial activity, cytotoxicity and toxicology. The conclusions in this paper provided an experimental and theorical foundation for its practical application. However, the fiber was failed to produce into fabric such as hydroentangle nonwovens due to the limitation of our experimental equipment, and thus the manufabrication and properties of fabrics need to be further studied in future.
本课题通过压力差作用和银镜反应制备得到具有抗菌性能的中空载银纤维——银纤维。初生态银粒子被置换出来而附着于纤维中空内表面层。通过SEM观察到银粒子的尺寸大约为0.1～0.5μm,且均匀地分布于纤维中空内表面层中。X-衍射分析观察到衍射峰只在20=38.2°,44.3°和64.5°处出现,这也证实了银粒子的存在以及银粒子的尺寸还没能大到使其衍射峰出现在2θ=77.4°和81.5°处。而这种尺寸的初生态银粒子具有极强的活性而使得该材料具有更强的抗菌性能。
银离子的释放是载银抗菌材料具有抗菌性的最基本条件。本课题制备的中空载银纤维中的银离子是通过纤维头尾两端释放。通过对银离子释放试验及其定性分析,研究了影响银纤维中银离子释放的因素,包括释放时间、纤维长度、银粒子含量以及纤维种类等。随着时间的增加,释放的银离子浓度逐渐增加并于3天后达到平衡;当纤维重量一定时,随着纤维的长度的增加,纤维根数减少,即能够释放银离子的头尾两端减少,因此银离子释放减少;银离子的释放量随着银粒子含量的增加而增加;不同种类的纤维其吸湿性不同,吸湿性较好的其银离子释放量也较多。
作为医用材料来说,银离子浓度是一个极为重要的参数。如果银离子浓度太高,就会对人体组织产生影响,如“银中毒”;如果太低,抗菌作用则受到限制。因此确定银离子浓度的阀值至关重要。本课题在Lotka-volterra模型的基础之上,建立银离子-细菌模型,确定了银离子浓度阈值,(?),其中,a值因菌种不同而不同,本文中金黄色葡萄球菌的a=0.82;λ值则与中空载银纤维的结构参数有关。这两个数据均可通过实验容易获得,从而计算得到银离子浓度阙值。而银纤维的抗菌性能试验表明银纤维具有广谱抗菌性及抗耐药菌性。影响抗菌效果的因素包括有:外界环境潮湿与否、纤维量及纤维结构参数等。当银纤维浓度为37.5μg/ml时,抗菌效果将达到90%以上。通过银纤维处理前后金黄色葡萄球菌和大肠埃希菌的SEM照片可以看出,银纤维处理后的细菌膨胀至崩裂,而且还发现银纤维对金黄色葡萄球菌的破坏作用大于大肠埃希菌,这可能是由于它们的细胞壁组成不同而造成的。
为了探讨中空载银纤维的细胞安全性,本课题选用了三种传代人系细胞株(Vero、Hela和WI-38)和一种原代细胞(从小儿包皮提取的上皮细胞)作为试验用人系细胞株进行安全性试验。试验结果表明,在银纤维浓度不超过4mg/ml时(包括抗菌浓度37.5μg/ml),银纤维的加入对人系细胞株的存活。而从电镜图片观察到,当加入银纤维达到4mg/ml时,细胞开始出现圆缩、凹陷等现象,这些现象可能是细胞死亡的一种前兆。
本课题还从免疫学和卫生毒理学的角度,通过皮肤覆盖和皮下埋植两种途径,检测了中空载银纤维对豚鼠的免疫水平(包括体液免疫、细胞免疫和非特异性免疫)和遗传物质的影响,并对主要器官,如心、肝、脾、肺、肾进行病理分析。通过对银纤维对豚鼠的免疫功能和遗传物质的影响的初步研究发现,当皮肤覆盖或埋植少于0.03g银纤维时,对免疫功能、遗传物质和主要器官都未造成损伤。但是当埋植高剂量银纤维,如0.03g时,溶血空斑细胞、循环免疫复合物和红细胞脆性都受到了影响,且病理切片分析也证实了埋植0.03g银纤维后,肝脏和肾脏开始出现病变。
综上所述,本课题通过压力差作用和银镜反应制备得到了抗菌中空载银纤维,并对银纤维的释放性能、抗菌性、细胞安全性及豚鼠毒理性进行了研究,并得出了相关结论,为该材料的实际应用提供了实验及理论基础。研究表明,该中空载银纤维具有很好的抗菌效果,当银纤维浓度达到37.5μm/ml时,抗菌效果就已经达到90%以上;当银纤维浓度在4mg/ml范围内时(包括抑菌浓度37.5μg/ml),银纤维的加入对人系细胞株的存活和形态均无影响;而当银纤维覆盖于豚鼠皮肤或埋植少于0.03g银纤维于豚鼠皮下时,银纤维的加入都不影响豚鼠的免疫功能、遗传物质及器官组织。但是当埋植超过0.03g银纤维后,造成了人系细胞株及豚鼠机体的轻微损伤。因此,我们在使用过程中应注意加强对载银材料的管理,尽快建立和完善载银材料安全性的研究方法和体系,重视载银材料的剂量-效应关系,为载银材料的应用提供可靠的依据。同时由于条件的限制,本课题未能将该材料制备成纺织产品,例如水刺无纺布,并进行性能研究,是本课题的不足之处,还有待于后续研究进一步完善。
Silver has been used for centuries to prevent and treat a variety of diseases, mostly notably infections. Previous work has shown that silver ions have strong inhibitory and bacterial effects as well as broad spectrum of activity, and concomitant low toxicity to mammalian cells. The inhibitory effect of silver ions was believed to be due to its sorption to the negatively charged bacterial cell wall, deactivating cellular enzymes, disrupting membrane permeability, and ultimately leading to cell lysis and death. Nowadays the introduction of new silver-based antibacterial polymers represented a great challenge for both academic world and industry. The silver-based thermoplastic polymer composites combined the excellent process properties of the thermoplastics with the inherent antibacterial ability of the silver. The products loaded with silver had disadvantages more or less, as lasting, staining, uncontrolled release, and et al.
Hollow fiber loaded with silver particles (silver fiber) was prepared in the study through the different press effect and the silver mirror reaction, and the silver was displaced and adhered on the fiber inner wall. The size of silver particles was approximately 0.1～0.5μm, which was measured by SEM test. X-ray of PET hollow fiber was compared to the silver fiber. The most important peaks are denoted clearly. The curve exhibited the obvious peaks at 2θ=38.2°, 44.3°, and 64.5°, which are due to the face-centered cubic crystalline silver, corresponding to the crystal faces of (111), (200), and (220) of silver, respectively. And the peaks at 2θ=77.4°and 81.5°, which are due to the crystal faces of (311) and (222) of silver, had not appeared. Therefore, it was very clear that the silver fiber was consisted of silver particles and the size of silver particles was small, which was consistent with SEM results.
When the silver fiber was entered in a water environment, the silver ions would be released form the two ends of the fiber. And the silver ions were the principle factor on the antibacterial efficiency. So the release of silver ions was investigated by release experiments and qualitative analysis. It was found that the concentration of silver ions released from the silver fiber was affected by many factors, such as release hours, fiber length, concentration of silver particles and fiber type. With the increase of release hours, the concentration of silver ions released from the silver fiber increased, and would be invariable after 3 days. And when there was the same weight of silver fiber, with the increase of the length of silver fiber, there were fewer points to release silver ions. And the silver ions released increased as well as the increase of concentration of silver particles. The study also indicated that the hollow fiber which had good moisture absorption released more concentration of silver ions.
In this paper, a silver ion-bacterium model was established to determine the threshold concentration of silver ions. The silver ion-bacterium model was similar to predator-prey model also known as the Lotka-Volterra model which described the dynamics of biology system in which two species interacted, one a predator and one its prey. Silver ions were antibacterial and treated as predators; bacteria were killed by silver ions and treated as preys. The concentration of silver ions was a critical parameter in medicine applications. When it was too high, it was lethal to the human organism, this was Argyria; while it was too low, the inhibitory efficiency was seriously affected. It was important to determine the threshold concentration of silver ions, , where a varied with different bacteria, andα=0.82 for S. aureus;λvaried with different hollow fibers and number of silver particles on the inner surface. They were easy to be got by experimental results.The antibacterial test showed that the silver fiber had great board-spectrum antibacterial activity, and had inhibitory effect on drug resistant bacteria. The antibacterial efficiency was dependent on the wateriness of the fibers, because the watery silver fiber released silver ions more than that of waterless. The antibacterial concentration of 38mm silver fiber was 37.5μg/ml. At that concentration, the inhibitory ratio reached up to 90%. From the SEM images of bacteria treated by silver fiber, it was found that the bacteria was expanded and cracked, and the different effect on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) may be due to the different configuration of cell walls of these two bacteria.
Four clay cell types were chosen to determine the cytotoxicity of the silver fiber, which were Vero, Hela, WI-38 and fibroblasts developed from primary culture of discarded surgical tissue, foreskins. And the experimental results showed that the cell proliferation was not affected by the concentration of silver fiber for antibacterial activity. And when treated by 4mg/ml silver fiber, the cell became less polyhedral, more fusiform, shrunken and rounded. It showed that cell proliferation and morphology of all cells was normal in the presence of the silver fiber down to a concentration of 4mg/ml, including 37.5μg/ml. However, abnormal size, shrinkage and rounded appearance of cells at higher dose (4mg/ml) suggested toxicity of the silver fiber.
During the process of the study, toxicity test on Guinea pig was conducted from the perspective of immunology and hygienic toxicology, in which two different contamination methods were used, including skin smearing and embedding. The effect of the silver fiber on immunity level, including humoral, cellular and non-special immunity, and germ plasm of Guinea pig were detected. Furthermore, pathological analysis was carried out on some primary organs, such as stomach, liver and kidney. Based on the elementary research on the effect of the silver fiber on immune function and germ plasm of the tested animals, it was found that in the presence of the silver fiber down to 0.03g, the silver fiber would not cause harm to the immune function, germ plasm and organs. But when in high dose, as 0.03g, hemolytie plaque-forming cell, immune complexes and red cell osmotic fragility was abnormal, and the pathological analysis further testified that the liver and kidney were really injured by 0.03g silver fiber. Therefore, the reasonable quantity control of the silver fiber during its application process is necessary, which requires that series of large-scale experiments need to be undertaken in further research process so as to acquire the dose-effect relation of silver fiber and provide reliable evidence for its extensive application.
The study prepared the hollow fiber loaded with silver particles and investigated its release property, antibacterial activity, cytotoxicity and toxicology. The conclusions in this paper provided an experimental and theorical foundation for its practical application. However, the fiber was failed to produce into fabric such as hydroentangle nonwovens due to the limitation of our experimental equipment, and thus the manufabrication and properties of fabrics need to be further studied in future.
引文
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