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This study aimed to assess the robustness of using a spray drying approach and formulation design in producing inhalable phage powders. Two types of Pseudomonas phages, PEV2 (Podovirus) and PEV40 (Myovirus) in two formulations containing different amounts of trehalose (70% and 60%) and leucine (30% and 40%) were studied. Most of the surface of the produced powders was found to be covered in crystalline leucine. The powders were stored at 4 °C and 20 °C under vacuum. The phage stability and in vitro aerosol performance of the phage powders were examined on the day of production and after 1, 3 and 12 months of storage. A minor titer loss during production was observed for both phages (0.2–0.8 log10 pfu/ml). The storage stability of the produced phage powders was found to be phage and formulation dependent. No further reduction in titer occurred for PEV2 powders stored at 4 °C across the study. The formulation containing 30% leucine maintained the viability of PEV2 at 20 °C, while the formulation containing 40% leucine gradually lost titer over time with a storage reduction of ∼0.9 log10 pfu/ml measured after 12 months. In comparison, the PEV40 phage powders generally had a ∼ 0.5 log10 pfu/ml loss upon storage regardless of temperature. When aerosolized, the total in vitro lung doses of PEV2 were of the order of 107 pfu, except the formulation containing 40% leucine stored at 20 °C which had a lower lung dose. The PEV40 powders also had lung doses of 106–107 pfu. The results demonstrate that spray dried Myoviridae and Podoviridae phage in a simple formulation of leucine and trehalose can be successfully stored for one year at 4 °C and 20 °C with vacuum packaging. The University of Sydney; Australian Research Council; National Institute of Allergy and Infectious Diseases of the National Institutes of Health; National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows reproducible mixing in miliseconds on the nanoliter scale. Here we investigated the feasibility of a microfluidic flow focusing setup built from commercially available fittings to encapsulate phages into liposomes. Two types of Pseudomonas phages, PEV2 (Podovirus, ~65 nm) and PEV40 (Myovirus, ~220 nm), were used as model phages. A mixture of soy phosphatidylcholine and cholesterol at a ratio of 4:1 dissolved in absolute ethanol with a total solid content of 17.5 mg/mL was injected through the center inlet channel of a cross mixer. Phage suspensions were injected into the cross mixer from the two side channels intersecting with the center channel. The total flow rate (TFR) varied 160 – 320 µL/min and the organic/aqueous flow rate ratio (FRR) varied 1:3 to 2:3. The size of liposomes and the encapsulation efficiency both increased with increasing FRR and slightly decreased with increasing TFR. Due to the different size of the two studied phages, the size of liposomes encapsulating PEV2 were smaller (135 – 218 nm) than those encapsulating the Myovirus PEV40 (261 – 448 nm). Highest encapsulation efficiency of PEV2 (59%) and PEV40 (50%) was achieved at a TFR of 160 µL/ml and a FRR of 2:3. Generally, the encapsulation efficiency was slightly higher than that obtained from the conventional thin film hydration followed by extrusion method. In summary, the proposed microfluidic technique was capable of encapsulating phages of different size into liposomes with reasonable encapsulation efficiency and minimal titer reduction. This work was financially supported by the Australian Research Council (Discovery Project DP150103953). SSY Leung is a research fellow supported by the University of Sydney. WJ Britton is funded by the National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225). The authors wish to acknowledge the technical support of Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis, The University of Sydney.

Phage therapy is a possible alternative to conventional antibiotics to treat pulmonary infections caused by multidrug resistance (MDR) bacterial strains [1-4], due to its high specificity, low toxicity, capability of auto-dosing and biofilm penetration. However, most research has been confined to liquid presentations using intranasal instillation and nebulization. Therefore, there is an aim to develop efficacious and stable phage powder formulations for easy storage, transport and administration. We have previously demonstrated the suitability of spray drying and spray freeze drying to incorporate phages into inhalable dry powders [5], and shown the effect of storage humidity on the long term stability of the spray dried phage powders [6]. In the present work, we extend this to investigate the effect of storage temperature and leucine on the long term stability of inhalable powders of two types of Pseudomonas phages, PEV2 and PEV40. This work was financially supported by the Australian Research Council (Discovery Project DP150103953). SSY Leung is a research fellow supported by the University of Sydney. WJ Britton is funded by the National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225).