Advanced Drug Delivery Group, Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, NSW 2006, Australia.
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Int J Pharm. 2023 Nov 5;646:123505. doi: 10.1016/j.ijpharm.2023.123505. Epub 2023 Oct 11.
Bacteriophages or phages used as an alternative therapy for treating multi-drug resistant infections require formulation consideration. Current strategies to produce phage formulations involving organic solvents are based on empirical practices without a good understanding of phage stability during formulation development. In this study, we investigated the effect of common formulation organic solvents (ethanol, isopropyl alcohol, tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO)) on the stability of Pseudomonas aeruginosa-specific myovirus (PEV1, PEV20) and podovirus (PEV31) phages using biological assay, transmission electron microscopy (TEM) and scattering near field optical microscopy (SNOM). The three phages were mixed with the solvents at different concentrations (25%, 50%, and 75% (v/v)) for 20 min. All phages were fully viable in the organic solvents at 25% (v/v) showing negligible titre changes. At the higher solvent concentration of 50% (v/v), the myoviruses PEV1 and PEV20 remained relatively stable (titre loss 0.4-1.3 log), whereas the podovirus PEV31 became less stable (titre loss 0.25-3.8 log), depending on the solvent used. Increasing the solvent level to 75% (v/v) caused increased morphological changes in TEM and decreased viability as indicated by the titre loss (0.32-7.4 log), with DMSO being the most phage-destabilising solvent. SNOM spectra showed differences in the signal intensity and peak positions in the amide I and amide II regions, revealing altered phage proteins by the solvents. In conclusion, the choice of the solvents for phage formulation depends on both the phages and solvent types. Our results showed (1) the phages are more stable in the alcohols than DMSO and THF, and (2) the myoviruses tend to be more stable than the podovirus in the solvents. Overall, a low to moderate (25-50 % v/v) level of organic solvents (except 50% THF) can be used in formulation of the phages without a substantial titre loss.
噬菌体或噬菌体作为治疗多重耐药感染的替代疗法，需要考虑制剂的问题。目前生产噬菌体制剂的策略涉及有机溶剂，这些策略基于经验实践，而对制剂开发过程中噬菌体的稳定性缺乏很好的了解。在这项研究中，我们使用生物测定法、透射电子显微镜（TEM）和近场散射光学显微镜（SNOM）研究了常见制剂有机溶剂（乙醇、异丙醇、四氢呋喃（THF）和二甲基亚砜（DMSO））对铜绿假单胞菌特异性肌尾噬菌体（PEV1、PEV20）和短尾噬菌体（PEV31）噬菌体稳定性的影响。将三种噬菌体分别与溶剂以不同浓度（25%、50%和 75%（v/v））混合 20 分钟。在 25%（v/v）的有机溶剂中，所有噬菌体均完全有活性，其效价变化可忽略不计。在更高的溶剂浓度 50%（v/v）下，肌尾噬菌体 PEV1 和 PEV20 相对稳定（效价损失 0.4-1.3 log），而短尾噬菌体 PEV31 则变得不太稳定（效价损失 0.25-3.8 log），这取决于所用的溶剂。将溶剂水平提高到 75%（v/v）时，TEM 中观察到形态变化增加，效价损失表明存活率降低（0.32-7.4 log），其中 DMSO 是最破坏噬菌体的溶剂。SNOM 光谱显示酰胺 I 和酰胺 II 区域的信号强度和峰位置存在差异，表明溶剂改变了噬菌体蛋白。总之，噬菌体制剂的溶剂选择取决于噬菌体和溶剂的类型。我们的结果表明：（1）噬菌体在醇类中的稳定性高于 DMSO 和 THF；（2）在溶剂中，肌尾噬菌体比短尾噬菌体更稳定。总体而言，低至高（25-50%v/v）浓度的有机溶剂（50%THF 除外）可用于噬菌体制剂，而不会导致显著的效价损失。
