Silver nanoparticles and anti-bacterial silver coating: research and development

doi:10.3969/j.issn.2095-4344.2016.25.022

Abstract

Abstract:

BACKGROUND: Current numerous studies have confirmed that silver nanoparticles have been extensively applied due to their good anti-bacterial performances.

OBJECTIVE: To summarize the overseas and domestic research and development of anti-bacterial silver coating based on the anti-bacterial mechanism and bio-safety of silver nanoparticles as well as progression of anti-bacterial silver coating.

METHODS: The first author retrieved the databases of Web of Science, PubMed and CNKI for relative articles published from January 1976 to January 2015. The keywords were “silver nanoparticles, titanium alloy, implant, antibacterial properties, biosecurity, coating” in English and Chinese, respectively.

RESULTS AND CONCLUSION: Because of their small dimension and quantum effects, as well as great specific surface area, silver nanoparticles can be in close contact with pathogenic microorganisms to exert biological effects. Additionally, silver nanoparticles exhibit the excellent resistance to bacteria, fungi, viruses and cancer cells. However, most of their mechanisms of biological effects remain unclear, and there are unresolved problems about the slow-release time of silver nanoparticles in vivo and how to control the slow-release silver ions. Besides, there are fewer long-term animal experiments. Therefore, a large number of laboratory and clinical studies are needed to ensure that silver nanoparticles cannot cause adverse reactions during long-term administration and how to reduce their toxicity.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Nanoparticles, Silver, Tissue Engineering

CLC Number:

R318

Xu Lian-chun, Shang Jian, Sun Ye, Han Xin-guang, Liu Zhen. Silver nanoparticles and anti-bacterial silver coating: research and development[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(25): 3793-3900.

Figures/Tables 1

2.1 银离子抗菌剂的应用随着对银抗菌能力的逐渐认识，其在预防及治疗感染当中扮演了重要角色[6]。有学者研究金属离子的抗菌性能，按其强弱顺序排列如下：Ag>Co>Ni≥Al≥ZnCu=Fe>Mn≥Sn>Ba≥Mg≥Ca[7]，对人体安全性顺序如下：Ag>Co>Ni>A1> Zn> Cu=Fe>Mn>Sn>Ba>Mg>Ca[8]。因为汞、铬、镉等部分金属离子对人体有害，因此实际上用作抗菌涂层的金属离子主要是银、铜和锌，其中银离子因强力的抗菌效果、良好的热稳定性、持久的抗菌效果，并对细胞及组织无明显毒性[9-10]，受到众多学者的关注。另有国外研究显示，银纳米颗粒在45 nm以下且浓度大于31.75 µg/g时，才会有明显的细胞毒性[11]。也有研究显示，只要银离子浓度达到0.1 µg/g就可以发挥抗菌作用，因此只要控制好银离子抗菌剂剂量，银离子十分适合作为制备生物材料抗菌涂层[12]。目前载银纳米材料已被广泛应用于医疗卫生领域，如医用敷料、外科器材、义肢等医疗器械[13-16]。 2.2 银纳米颗粒抗菌机制研究银离子作为优良的抗菌剂，有着广谱的抗菌范围，可抑制阳性革兰阴性、革兰阳性菌、真菌、原生动物甚至病毒[17]。根据目前国内外的研究成果，银离子的杀菌机制有以下几种可能：①抑制细菌细胞壁表面肽聚糖的合成，破坏细胞壁完整性，使其失去对细菌的保护作用，导致细菌死亡；破坏细菌的细胞膜，影响其呼吸代谢功能，使其细胞内容物丢失死亡。有研究表明，银纳米颗粒可穿过细胞壁后聚集在细胞膜表面，干扰细胞膜的连续性，并能够大幅度扰乱其透气性和呼吸，甚至导致细胞死亡[18-22]。银纳米颗粒还可与细菌外膜上的脂质结合，抑制耐药菌的感染[23]。②影响酶的活性，破坏蛋白质的合成，阻止细菌的生长与繁殖；干扰DNA、RNA合成，阻止遗传信息的转录复制，使细菌无法繁殖。银离子进入细菌内与其蛋白质中的巯基(-SH)、氨基(-NH2)基团反应，阻止蛋白质和酶的合成[24]。银离子作用于细菌时，还可通过核膜进入细胞核内，改变松散的DNA结构，导致DNA团聚，形成团块，影响DNA复制及转录[25]。③银纳米颗粒在被氧化过程中可以产生活性氧自由基，其可攻击生命大分子物质及细胞壁，使类脂中的不饱和脂肪酸发生过氧化，导致细胞膜结构破坏，并能够氧化蛋白质，具有广泛杀灭微生物的作用，包括细菌、芽胞、病毒、真菌等。有研究指出，银纳米颗粒可催化活性中心，激发产生电子，将银离子所吸附的氧分子还原成超氧化物自由基[26]。而激发失去电子的正电荷的基团，可以使氢氧根阴离子氧化，形成羟基自由基。这些自由基团可抑制细菌的增殖，导致细菌迅速死亡，达到抗菌目的。④银纳米颗粒的粒径大小是影响其抗菌性的重要因素。多项研究证明，小粒径能够明显增加银纳米颗粒的比表面积，能更有效释放银离子。因此粒径的大小与银离子浓度及量相比更加有意义[27]。 2.3 银纳米颗粒研究进展银纳米颗粒由于其特有的抗菌机制，在防治细菌、真菌、病毒及癌症方面亦可表现其生物效应。 2.3.1 银纳米颗粒在防治细菌中的应用 Devi等[28]的研究发现，银纳米颗粒对金黄色葡萄球菌、酿脓链球菌、沙门氏菌和粪肠球菌有良好的抗菌功效。此外，对于抑制克雷伯氏菌、肺炎球菌和肠杆菌，银纳米颗粒与红霉素、甲氧西林、氯霉素和环丙沙星在抗菌上有优异的协同效应[29]。Shameli等[30]通过琼脂扩散方法验证了聚乙二醇-银纳米颗粒对革兰阳性菌和革兰阴性菌良好的抑菌性，还证明通过控制纳米粒子的大小可显著抑制这些病原体增长。因此在制备银纳米颗粒抗菌涂层中，充分利用银纳米颗粒的大小及与抗生素的协同作用，对于防治内植物细菌感染有着显著作用。 2.3.2 银纳米颗粒在抗真菌活性中的作用银纳米颗粒在抗真菌方面的研究较少。Kim等[31]证明化学方法合成的银纳米颗粒材料，在抗白色念球菌、假丝酵母菌及须癣毛癣菌有良好的杀菌活性。Gajbhiye等[32]报道生物合成方法的银纳米颗粒对抗茎点霉、半裸镰刀菌、木霉菌属和白色念珠菌有抑制作用。此外，他们认为银纳米材料与氟康唑协同作用效果更显著。Kaur等[33]研究证实，银和壳聚糖纳米颗粒结合对抗立枯丝核菌、链格孢菌有潜在的活性。Tile等[34]报道银纳米颗粒对抗白色念珠菌、红毛癣菌和烟曲霉菌有明显的抗菌效果。尽管目前众多文献对于银纳米颗粒抗真菌机制没有明确的报道，但其中的一种机制可能是银纳米颗粒对于真菌质膜的破坏及抑制真菌的出芽过程[35]。 2.3.3 银纳米颗粒的抗病毒作用文献证明银纳米颗粒的抗菌性能已受到极大的关注，但在抗病毒方面仍然是一个未得以发展的研究领域[36]。如今，安全有效的抗病毒治疗可显著改善大量患者的生活质量。理想的抗病毒制剂应具有广谱作用，尤其对于不可预见的病毒流行或大流行应用。前根据文献推测，银纳米颗粒抗病毒特性主要表现在3方面：在抗原核生物、真核生物病毒方面，有实验研究银纳米颗粒有着较广的抗病毒特性[37]；Lara等[38-39]研究发现抗病毒活性依赖银纳米颗粒的大小(一般情况下银纳米颗粒小于 25 nm，其抗病毒活性明显增加)；银纳米颗粒可影响病毒的复制周期[40-41]。Gaikwad等[42]研究发现，银纳米颗粒对于人副流感病毒3型及单纯性疱疹病毒1型、2型有良好的抗病毒性能。Salem等[43-44]研究发现，对于抗柯萨奇病毒B3、猴天花银纳米颗粒亦表现出优良的活性。另外有研究发现，银纳米颗粒对于HIV-1、HSV、RSV可表现出抗病毒特性[45]。目前银纳米颗粒作为抗病毒药物的开发处于起步阶段，仍需进一步的研究才能阐明机制。 2.3.4 银纳米颗粒的抑制癌细胞作用有研究发现，利用基因生物合成的银纳米颗粒，可使人乳腺癌细胞发生氧化应激，破坏了细胞的完整性，加速细胞凋亡[46]。基因生物合成的银纳米颗粒可抑制人类结肠癌中HCT15细胞系的增殖，此结果表明，银纳米颗粒可能对结肠癌细胞系起到抗增殖作用，抑制其增长，阻止G0 /G1期，减少DNA合成和诱导细胞凋亡[47]。尽管诸多实验研究证明银纳米颗粒在细菌、真菌、病毒及癌症方面均表现出一定有效的活性，究其原因与银纳米颗粒自身粒径大小、剂量、作用时间及其固有属性密不可分，但其制备方法及外界条件对银纳米颗粒诸多特性有着一定的影响，所以仍需大量的体内外研究。 2.4 银纳米颗粒生物安全性能研究银纳米颗粒具有良好的抗菌作用，但同时也可能会对人体正常细胞产生不良反应。研究证明，银纳米颗粒可诱导斑马鱼胚胎细胞畸形发育[48]，破坏细胞膜[49]，诱导产生遗传毒素和细胞毒素，损害人类肺纤维组织及胶质瘤细胞[50]，甚至抑制人类免疫系统[51]。因此对于银纳米颗粒生物安全性的研究是极其重要的。但目前银纳米颗粒的生物安全性存在较多的争议，其毒性机制尚不清楚。国内有学者应用载银凝胶制剂对小鼠进行急性毒性实验，发现载银凝胶制剂仅对家兔阴道黏膜有极轻度刺激，而皮肤变态反应实验、皮肤刺激实验、骨髓嗜红细胞微核实验等均为阴性结果，证明载银凝胶制剂具有一定的生物安全性[52]。Pauksch等[53]研究发现，银纳米颗粒主要影响人类间充质干细胞及成骨细胞，并且当银纳米颗粒浓度达10 µg/g或更高时有细胞毒性。因而认为载银纳米医疗产品也许会存在一个良好的治疗窗，可应用于临床。Necula等[54]于体外检测银纳米颗粒的细胞毒性，实验结果发现高浓度含量的银纳米颗粒(3.0 Ag)具有极强的细胞毒性，相对浓度较低的银纳米(0.3 Ag)不仅成骨细胞生长良好，并且表现良好的抗菌性能，证明了银纳米细胞毒性不仅取决于量的多少，还取决于银纳米颗粒粒径大小。相关研究从影响细胞DNA合成方面研究银纳米颗粒的细胞毒性。Kovvurual等[55]发现，口服银纳米颗粒可诱导大鼠DNA损害并可永久导致基因变异，他们证实在胚胎中银纳米颗粒致使DNA节段缺失。在骨髓中染色体不可逆转改变，在外周血中DNA双链断裂，DNA氧化损伤。这些问题反映了银纳米颗粒使用的安全性，尤其是高浓度银纳米颗粒粒子的释放。尽管在人类尚未有实验证明银纳米颗粒对人体的损害，需要进一步的大量研究去验证。 2.5 生物材料载银涂层的研究进展内植物的感染也称为“生物材料相关感染”，文献报道指出钛合金牙种植体钉道感染需住院进行抗生素治疗，取出螺钉或拆除外固定支架所发生的严重钉道感染，其感染率达5.8%[56]，眼旁路置换感染率4%-30%，在骨科中，全肘关节置换术后感染率可高达4%-47%[57]。为了减少感染的发生，减少抗生素使用带来的影响，在生物材料中加入抗菌涂层是一种有效可行的方法[58]。随着医疗技术的进步和生活水平的不断提高，钛合金因其优良的力学性能、强度-质量比、耐腐蚀性及生物相容性，已被广泛应用于临床[59]。目前载银钛合金抗菌涂层制备方法主要分为3大类：物理方法、化学方法及生物方法。物理方法主要有蒸发凝聚法、离子溅射法、热喷涂、真空管蒸发镀和阳极氧化沉积及等离子注入等方法。阮洪江等[60]通过等离子热喷涂方法制备了载银羟基磷灰石材料，实验证明所得载银涂层具有良好的生物安全性、生物相容性和较强的抗菌性能。王宇华等[61]通过真空等离子喷涂技术，在钛基材表面制备出载银羟基磷灰石涂层材料，实验证明当单质银质量百分比>3%时，实验样品有抑菌作用。化学方法主要有高温分解法[62]、电化学镀法[63]、光化学镀法[64]、声化学方法和表面还原法等方法[65-66]。最近出现了生物制备纳米银材料方法，这一发现消除了化学方法带来的化学毒性，同时增加了银纳米颗粒的生物相容性[67]。生物方法合成载银纳米材料主要依靠微生物的酶催化机制和微生物自身的基团氧化还原反应，微生物产生的酶，作为电子传递体使氢气、甲酸盐等还原性物质的电子传递给金属离子，从而被还原。目前大量的生物有机体被使用于制备银纳米颗粒材料中，包括真菌、细菌及微生物等。一项研究使用金黄色葡萄球菌还原银制备了微生物涂层材料，证明其可产生较高的银纳米颗粒，对耐甲氧西林表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌及化脓链球菌有良好的抗菌性能[68]。以上3种制备载银抗菌涂层材料的方法各有优缺点，需要大量实验分别验证其在体内的使用价值。 2.6 载银内植物在骨科中的应用现状银纳米颗粒作为抗菌剂在医学领域临床中已得到广泛应用，在骨科中应用尚未得到开展。国外文献报道，在假体周围感染、外固定针、骨髓炎伴骨折不愈合、骨水泥、敷料中，银纳米颗粒可部分应用，并收到较好的治疗效果。文献报道假体周围感染率可达9%-29%[69-71]。Hardes等[72]研究发现，载银组假体假体周围感染率(7%)明显低于普通假体感染率(47%)，并且没有明显的细胞毒性。针道感染在外固定器使用中是最常见的并发症，报道称其感染率可达42%[73]。Collinge等[74]对比载银涂层外固定针与不锈钢针的效果，证实载银涂层外固定针道感染率显著降低。在骨髓炎伴骨折不愈合中，由骨发生蛋白2与聚乳酸银合成的复合移植骨材料，更能刺激新骨形成[75]。Alt等[76]在体外对比将载银骨水泥、在庆大霉素骨水泥及普通骨水泥的抗菌效果，发现每一种骨水泥材料对表皮葡萄球菌、MRSE及MRSA均具有不同程度的抗菌效果，只有载银骨水泥对所有细菌具有强的抗菌性能。在抗菌敷料方面，银制品抗菌敷料已使用一段时间，最近含银纳米颗粒的敷料越来越受到欢迎。Wilkinson等[77]研究发现，载有银纳米颗粒敷料具有较好的抗菌效果，但其细胞毒性尚不明确。在载银涂层创伤内植物方面，其体外实验已得到了明显效果，但体内研究结果不尽相同[78-79]。Kose等[80]制备了一种载银钛合金内置物，结果证明其具有较高的抗菌性且较低的骨髓炎发生率。"

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