Antimicrobial peptides (AMP) are powerful components of the innate immune system, as they display wide activity spectrum and low tendency to induce pathogen resistance. Hence, the development of AMP-based coatings is a very promising strategy to prevent biomaterials-associated infections. This work aims to investigate if Dhvar-5-chitosan conjugates, previously synthesized by us via azide-alkyne "click" reaction, can be applied as antimicrobial coatings. Ultrathin coatings were prepared by spin coater after dissolving Dhvar-5-chitosan conjugate powder in aqueous acetic acid. Peptide orientation and exposure from the surface was confirmed by ellipsometry and contact angle measurements. Bactericidal activity was evaluated against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, the most prevalent pathogens in implant-associated infections. Results showed that Dhvar-5-chitosan coatings displayed bactericidal effect. Moreover, since Dhvar-5 has head-to-tail amphipathicity, it was clear that the bactericidal potency was dependent on which domain of the peptide (cationic or hydrophobic) was exposed. In this context, Dhvar-5 immobilized through its C-terminus (exposing its hydrophobic end) presented higher antimicrobial activity against Gram-positive bacteria and reduced adhesion of Gram-negative bacteria. This orientation-dependent antimicrobial activity was further corroborated by the anti-biofilm assay, as covalent immobilization of Dhvar-5 through its C-terminus provided anti-biofilm properties to the chitosan thin film. Immobilization of Dhvar-5 showed no cytotoxic effect against HFF-1 cells, as both metabolic activity and cell morphology were similar to control. In conclusion, Dhvar-5-chitosan coatings are promising antimicrobial surfaces without cytotoxic effects against human cells. STATEMENT OF SIGNIFICANCE: AMP-tethering onto ground biomaterial is still a poorly explored strategy in research. In this work, AMP-tethered ground chitosan is used to produce highly antibacterial ultrathin films. Powdered AMP-tethered chitosan appears as an alternative solution for antimicrobial devices production, as it is suitable for large scale production, being easier to handle for fabrication of different coatings and materials with antimicrobial properties and without inducing toxicity.

中文翻译：

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由Dhvar-5-click接枝的壳聚糖粉末制备的抗菌涂料。

抗菌肽（AMP）是先天免疫系统的强大组成部分，因为它们显示的活性谱宽，诱导病原体抗药性的趋势低。因此，基于AMP的涂层的开发是一种非常有希望的策略，可以防止与生物材料相关的感染。这项工作的目的是调查以前由我们通过叠氮化物-炔烃“点击”反应合成的Dhvar-5-壳聚糖共轭物是否可以用作抗微生物涂料。将Dhvar-5-壳聚糖共轭物粉末溶于乙酸水溶液后，通过旋涂机制备超薄涂层。肽的取向和从表面的暴露通过椭圆光度法和接触角测量得到证实。评估了对表皮葡萄球菌，金黄色葡萄球菌，大肠杆菌和铜绿假单胞菌的杀菌活性，植入物相关感染中最常见的病原体。结果表明，Dhvar-5-壳聚糖涂层具有杀菌作用。此外，由于Dhvar-5具有从头到尾的两亲性，因此很明显杀菌力取决于所暴露的肽（阳离子或疏水）结构域。在这种情况下，通过其C末端固定的Dhvar-5（暴露于其疏水端）对革兰氏阳性菌表现出更高的抗菌活性，并降低了革兰氏阴性菌的附着力。通过抗生物膜测定进一步证实了这种依赖于方向的抗菌活性，因为通过Dhvar-5的C端共价固定Dhvar-5可为壳聚糖薄膜提供抗生物膜特性。固定化Dhvar-5对HFF-1细胞没有细胞毒性作用，因为代谢活性和细胞形态均与对照相似。总之，Dhvar-5-chitosan涂层是有前途的抗菌表面，对人体细胞无细胞毒性作用。意义声明：在地面生物材料上进行AMP束缚仍是研究中探索不佳的策略。在这项工作中，使用AMP束缚的壳聚糖来生产高度抗菌的超薄薄膜。AMP束缚的壳聚糖粉可作为抗菌设备生产的替代解决方案，因为它适合大规模生产，更易于处理具有抗菌特性且不产生毒性的各种涂层和材料。Dhvar-5-chitosan涂料是有前途的抗菌表面，对人体细胞无细胞毒性作用。意义声明：在地面生物材料上进行AMP束缚仍是研究中探索不佳的策略。在这项工作中，使用AMP束缚的壳聚糖来生产高度抗菌的超薄薄膜。AMP束缚的壳聚糖粉可作为抗菌设备生产的替代解决方案，因为它适合大规模生产，更易于处理具有抗菌特性且不产生毒性的各种涂层和材料。Dhvar-5-chitosan涂料是有前途的抗菌表面，对人体细胞无细胞毒性作用。意义声明：在地面生物材料上进行AMP束缚仍是研究中探索不佳的策略。在这项工作中，使用AMP束缚的壳聚糖来生产高度抗菌的超薄薄膜。AMP束缚的壳聚糖粉可作为抗菌设备生产的替代解决方案，因为它适合大规模生产，更易于处理具有抗菌特性且不产生毒性的各种涂层和材料。