Microfluidic-assisted bacteriophage encapsulation into liposomes

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.