Abstract：
Methicillin-resistant Staphylococcus aureus (MRSA) biofilm-associated bacterial keratitis is highly intractable, with strong resistance to β-lactam antibiotics. Inhibiting the MRSA resistance gene mecR1 to downregulate penicillin-binding protein PBP2a has been implicated in the sensitization of β-lactam antibiotics to MRSA. However, oligonucleotide gene regulators struggle to penetrate dense biofilms, let alone achieve efficient gene regulation inside bacteria cells. Herein, an eye-drop system capable of penetrating biofilms and targeting bacteria for chemo-gene therapy in MRSA-caused bacterial keratitis is developed. This system employed rolling circle amplification to prepare DNA nanoflowers (DNFs) encoding MRSA-specific aptamers and mecR1 deoxyribozymes (DNAzymes). Subsequently, β-lactam antibiotic ampicillin (Amp) and zinc oxide (ZnO) nanoparticles are sequentially loaded into the DNFs (ZnO/Amp@DNFs). Upon application, ZnO on the surface of the nanosystem disrupts the dense structure of biofilm and fully exposes free bacteria. Later, bearing encoded aptamer, the nanoflower system is intensively endocytosed by bacteria, and releases DNAzyme under acidic conditions to cleave the mecR1 gene for PBP2a down-regulation, and ampicillin for efficient MRSA elimination. In vivo tests showed that the system effectively cleared bacterial and biofilm in the cornea, suppressed proinflammatory cytokines interleukin 1β （IL-1β） and tumor neocrosis factor-alpha （TNF-α）, and is safe for corneal epithelial cells. Overall, this design offers a promising approach for treating MRSA-induced keratitis.
摘要：
耐甲氧西林金黄色葡萄球菌（MRSA）生物膜相关细菌性角膜炎是高度难治的，对β-内酰胺类抗生素有较强的耐药性。抑制MRSA抗性基因mecR1下调青霉素结合蛋白PBP2a与β-内酰胺抗生素对MRSA的致敏有关。然而,寡核苷酸基因调节剂努力穿透致密的生物膜，更不用说在细菌细胞内实现有效的基因调控了。在这里，开发了一种能够穿透生物膜和靶向细菌的滴眼液系统，用于MRSA引起的细菌性角膜炎的化学基因治疗。该系统采用滚环扩增来制备编码MRSA特异性适体和mecR1脱氧核酶(DNAzymes)的DNA纳米花(DNF)。随后，将β-内酰胺抗生素氨苄青霉素(Amp)和氧化锌(ZnO)纳米颗粒依次加载到DNF(ZnO/Amp@DNF)中。一经申请,纳米系统表面的ZnO破坏生物膜的致密结构并充分暴露游离细菌。稍后，带有编码的适体，纳米花系统被细菌强烈内吞，并在酸性条件下释放DNA酶，以切割mecR1基因以下调PBP2a，和氨苄青霉素用于有效消除MRSA。体内试验表明，该系统有效清除了角膜中的细菌和生物膜，抑制促炎细胞因子白细胞介素1β（IL-1β）和肿瘤坏死因子α（TNF-α）,对角膜上皮细胞是安全的。总的来说,这种设计为治疗MRSA诱导的角膜炎提供了一种有希望的方法.
