Broad Spectrum Antiviral and Antibiofilm Materials to Treat Hospital Acquired Infections

全球每年有425万人死于急性呼吸道感染(市长，2010年)。支气管炎和肺炎等感染通常用抗生素治疗，使ARI成为抗菌素耐药性(AMR)的主要驱动力，而且鉴于抗菌素耐药性病原体的增加(O‘Neill，2016)，ARIS的治疗迫切需要抗生素的替代品。一氧化氮(NO)是免疫系统为应对入侵病原体而产生的一种有效的广谱抗菌剂。然而，它的反应性和较短的半衰期阻碍了其临床应用，使其在感染部位的传递往往具有挑战性。为了解决这个问题，没有开发出任何供体，如N-二氮杂二醇酸盐，以提高其稳定性和保质期。这种NO供体是由胺和NO气体在高压下反应形成的[3]，由于其易于制造，因此通常用于生物材料研究。明胶纳米粒(GNP)因其生物相容性、生物降解性和FDA批准而被用于许多药物输送系统。此外，由于明胶是一种多胺，它为N-重氮二醇酸盐的链结提供了多个结合部位。因此，我们制备了用于控制释放NO的明胶纳米粒(GNP/NO)，用于治疗ARI，并评价了其抗菌效果、生物相容性和NO释放动力学。采用两步去溶法合成了GNP，并通过动态光散射(DLS)和扫描电子显微镜(SEM)分析证实了其为均一的球状颗粒。通过傅立叶变换红外光谱(FT-IR)对N-重氮二醇酸盐纳米粒(GNP/NO)的物理化学性质进行了表征，证实了纳米粒子的功能化成功。用化学发光法测定了GNP/NO的有效载荷和释放动力学，结果表明GNP/NO在最初释放NO，随后持续释放超过24 h，并在24 h内对铜绿假单胞菌、大肠杆菌、金黄色葡萄球菌和白色念珠菌进行了抑菌效果测试，结果表明，GNP/NO在4 h和24 h后对所有受试微生物的活菌生长均有显著的抑制作用(最高可达3log)。随后，使用ISO-10993间接(渗滤液)方法评估了GNP/NO对L929和Detroit 562细胞系的细胞毒性，结果表明GNP/NO保持了70%以上的细胞存活率。我们已经成功地开发了GNP/NO，它在细胞相容范围内表现出良好的抗菌特性，说明GNP/NO作为治疗急性呼吸窘迫的潜力。在确保GNP/NO作为有效治疗ARIs的可行性之前，有必要进行进一步的研究，以评估GNP/NO的体内效应。急性呼吸道感染是世界第三大致死原因。《英国医学杂志》，2010年。341：P.C6360.2。《全球应对抗药性感染：最终报告和建议》。2016.3。通过与一氧化氮的厌氧反应，N-重氮二醇酸盐和N-亚硝胺的竞争性形成。org。让我们看看，2009年。11(23)。

Acute respiratory infections (ARIs) are responsible for 4.25 million deaths annually worldwide (Mayor, 2010). Infections such as bronchitis and pneumonia are commonly treated with antibiotics, making ARI a primary driver for antimicrobial resistance (AMR), and given the rise in antimicrobial-resistant pathogens (O'Neill, 2016), the treatment of ARIs necessitates a pressing need for alternatives to antibiotics. Nitric oxide (NO) is an effective broad-spectrum antimicrobial produced by the immune system in response to invading pathogens. However, its clinical application is hindered by its reactivity and short half-life, making its delivery to the site of infection often challenging. To address this issue, NO donors, such as N-diazeniumdiolates have been developed to improve its stability and shelf life. This NO donor is formed by the reaction of amines with NO gas under high pressure [3], and owing to its ease of fabrication, it is commonly used in biomaterial research. Gelatin nanoparticles (GNP) are used in many drug delivery systems owing to their biocompatibility, biodegradability and FDA approval. Furthermore, as gelatin is a polyamine, it provides multiple binding sites for the tethering of N-diazeniumdiolates. Therefore, we fabricated NO-loaded gelatin nanoparticles (GNP/NO) for the controlled release of NO for the treatment of ARI, and assessed their antimicrobial efficacy, biocompatibility, and NO release kinetics.A two-step desolvation method was used to synthesise GNP and produced homogenous spherical particles of ~225nm, confirmed by dynamic light scattering (DLS) and scanning electron microscopy (SEM) analysis. The physicochemical properties of the GNP loaded with N-diazeniumdiolates (GNP/NO) were assessed via Fourier-transform infrared spectroscopy (FT-IR), which confirmed the successful functionalisation of the nanoparticles. Chemiluminescence was used to determine the NO payload and release kinetics, demonstrating that GNP/NO released an initial NO burst, followed by a sustained release over 24 h. The antimicrobial efficacy of GNP/NO was tested against a panel of microorganisms, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans over a 24 h period and revealed a significant reduction (up to 3 log) in viable growth after 4 h and 24 h against all tested microorganisms. Following this, the ISO-10993 indirect (leachate) protocol was used to assess the cytotoxicity of GNP/NO against both the L929 and Detroit 562 cell lines, which showed that GNP/NO maintained over 70% cell viability.We have demonstrated the successful development of GNP/NO, which exhibits excellent antimicrobial properties within a cytocompatible range, illustrating the potential of GNP/NO as a treatment for ARI. Further investigation is warranted to assess the in vivo effects of GNP/NO before ensuring its feasibility for use as an effective treatment for ARIs.References1. Mayor, S., Acute respiratory infections are world's third leading cause of death. BMJ, 2010. 341: p. c6360.2. O'Neill, J., Tackling drug-resistant infections globally: final report and recommendations. 2016.3. Coneski, P.N., 'Competitive formation of N-diazeniumdiolates and N-nitrosamines via anerobic reactions with nitric oxide.' Org. Let., 2009. 11(23).