Advanced Drug Delivery Group, School of Pharmacy, University of Sydney, Sydney, NSW, Australia.
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA.
Int J Pharm. 2020 Dec 15;591:119982. doi: 10.1016/j.ijpharm.2020.119982. Epub 2020 Oct 14.
Endolysins are bacteriophage-derived protein molecules highly effective for bacterial killing. Cpl-1 and ClyJ-3 are native and chimeric endolysins, respectively, having antimicrobial activity against Streptococcus pneumoniae which causes lung infections. We conducted the first feasibility study on nebulisation of Cpl-1 and ClyJ-3, with a focus on the antimicrobial activity, structural changes of the proteins and aerosol performance. Bacterial colony counts, live cell imaging and Fourier-transform infrared(FTIR) spectroscopy were used to evaluate the proteins before and after jet or vibrating mesh nebulisation. These nebulised aerosols were inhalable with a volume median size of 3.8-4.2 µm (span 1.1-2.3) measured by laser diffraction. How-ever, neb-u-li-sa-tion caused al-most com-plete loss in bioac-tiv-ity of ClyJ-3, which were corroborated with the live cell imaging observation and protein structural damage with a large intensity reduction in the amide absorption bands between 1300 and 1700 cm. In contrast, the bactericidal activity of Cpl-1 showed no significant difference (p ≥ 0.05) before and after mesh nebulisation with 4.9 and 4.6-log10 bacterial count reduction, respectively. However, jet nebulisation reduced the bioactivity of Cpl-1 and the effect was time-dependent showing 1.7, 1.0-log10 bacterial count reduction at 7 and 14 min with complete loss of antimicrobial activity at 21 min after nebulisation, respectively. The results were consistent with time-dependent changes in live cell images and FTIR amide band changes at 1655, 1640, 1632 and 1548 cm. In conclusion, it is feasible to nebulise endolysins for inhalation delivery but it depends on both the protein and the nebuliser, with the mesh nebuliser being the preferred choice.
溶菌酶是一类高效的杀菌蛋白分子，来源于噬菌体。Cpl-1 和 ClyJ-3 分别为天然和嵌合溶菌酶，对引起肺部感染的肺炎链球菌具有抗菌活性。我们首次进行了雾化 Cpl-1 和 ClyJ-3 的可行性研究，重点研究了抗菌活性、蛋白质结构变化和气溶胶性能。使用细菌菌落计数、活细胞成像和傅里叶变换红外（FTIR）光谱法评估喷射或振动网孔雾化前后蛋白质的抗菌活性。这些雾化后的气溶胶可吸入，激光衍射测量的体积中位粒径为 3.8-4.2μm（跨度 1.1-2.3μm）。然而，雾化几乎完全导致 ClyJ-3 的生物活性丧失，活细胞成像观察和蛋白质结构损伤也证实了这一点，酰胺吸收带的强度在 1300-1700cm 之间显著降低。相比之下，Cpl-1 的杀菌活性在网孔雾化前后没有显著差异（p≥0.05），分别减少了 4.9 和 4.6-log10 个细菌数。然而，喷射雾化会降低 Cpl-1 的生物活性，且该效应具有时间依赖性，分别在 7 和 14 分钟时减少 1.7 和 1.0-log10 个细菌数，在雾化后 21 分钟时完全丧失抗菌活性。这些结果与活细胞图像和 FTIR 酰胺带在 1655、1640、1632 和 1548cm 处的时间依赖性变化一致。总之，雾化溶菌酶进行吸入给药是可行的，但这取决于蛋白质和雾化器，网孔雾化器是首选。
