Selectively Antibacterial Silver-Gold Alloy Nanoparticles Conjugated with Target Specific Aptamer Sequences

植入物相关感染发生在许多不同的医学学科中，并且是现代医学中最重要的挑战之一。这些感染是由组织在复杂生物膜中的细菌引起的，这些生物膜对抗生素和宿主的免疫系统具有高度抗性。这通常会导致植入物丢失，在某些学科中甚至会导致危及生命的并发症。抗菌植入物表面和活性物质已用于预防或治疗。然而，这些方法的缺点是不能选择性地和有效地处理细菌细胞而不对人类细胞造成长期损害。因此，有很大的需求，创新的抗感染策略，扩大治疗窗口。在本项目中，将开发针对特定病原菌的特异性活性的银-金合金纳米粒子（AgAu-NP）。这涉及将抗菌但也具有细胞毒性的Ag-NP和高度生物相容性的Au-NP的性质相结合。这里，银通过Ag+离子释放负责杀菌活性，而金意在增加颗粒稳定性，控制离子释放并允许通过金-硫醇化学与靶特异性适体缀合。为此，通过使用先进的基于激光的方法合成稳定的AgAu-NP，该方法允许合成具有受控组成的这些合金纳米颗粒，而所得的NP与硫醇化适体序列异位缀合。在这种情况下，颗粒组成（AgAu）和颗粒大小对金属离子释放，表面覆盖和共轭效率在生物共轭，以及形成可溶性和不溶性的银物种在不同的介质中的影响将系统地检查。由于这些AgAu-NP-缀合物与相关细菌如S.金黄色葡萄球菌和牙龈卟啉单胞菌，病原体暴露于在颗粒附近发现的高局部Ag+浓度，其选择性地抑制这种细菌的生长，即使在存在其它菌株的情况下。为了推导出理想的NP组成并实现最大的抗菌活性与最小的毒性相结合，将进行各种系统的分子生物学测定和细胞培养试验。这包括不同细菌菌株以及与人类细胞平行的相关细菌的共培养模型。选择性结合实验将探讨的AgAu-NP-共轭物的细菌的特定关联的可能性，而，此外，细胞摄取检查，以阐明杀菌活性的机制。计划中的实验提供了一种治疗细菌感染的高度创新的方法，并将阐明关于NP组合物如何影响生物缀合的基本科学知识。

Implant- associated infections occur in many different medical disciplines and are one of the most important challenges in modern medicine. These infections are caused by bacteria organised in complex biofilms which are highly resistant to antibiotics and to the host's immune system. This can often lead to loss of the implant and - in some disciplines - even to life-threatening complications. Antibacterial implant surfaces and active substances have been used for prophylaxis or therapy. However, these have the disadvantage that bacterial cells cannot be selectively and effectively treated without causing long-term damage to human cells too. Consequently, there is great need for innovative anti-infective strategies with a broadened therapeutic window.In this project, targeting silver-gold alloy nanoparticles (AgAu-NP) with specific activity against selected pathogenic bacteria will be developed. This involves combining the properties of antibacterial but also cytotoxic Ag-NP and highly biocompatible Au-NP. Here silver is responsible for the bactericidal activity by Ag+ ion release, whereas gold is meant to increase particle stability, to control ion release and to allow conjugation with target-specific aptamers via gold-thiol chemistry. To this end stable AgAu-NPs are synthesised by using an advanced laser-based method, which allows synthesis of these alloy nanoparticles with controlled composition, while the resulting NPs are conjugated ex situ with thiolated aptamer sequences. In this context the influence of particle composition (AgAu) and particle size on metal ion release, surface coverage and conjugation efficiency during bioconjugation as well as the formation of soluble and insoluble silver species in different media will be systematically examined. Due to specific binding of these AgAu-NP-conjugates to the bacterial membrane of relevant bacteria like S. aureus and P. gingivalis the pathogens are exposed to a high local Ag+ concentrations found in the vicinity of the particles which selectively inhibits the growth of this bacterium, even in the presence of other strains. In order to deduce the ideal NP composition and to achieve maximal antibacterial activity combined with minimal toxicity, a variety of systematic molecular biological assays and cell culture tests will be performed. This includes co-culture models of different bacteria strains as well as of relevant bacteria parallel to human cells. Selective binding experiments will explore the possibility of a specific association of the AgAu-NP-conjugates to the bacteria, while, furthermore, cellular uptake is examined in order to elucidate the mechanism of bactericidal activity. The planned experiments provide a highly innovative procedure for the treatment of bacterial infections and will elucidate basic scientific knowledge on how bioconjugation is affected by NP composition.