(M0930-06-34) Encapsulation of Bacteriophages in Liposomes for Improved Inhaled Delivery

Purpose: The alarming threat of multidrug-resistant bacteria in lung infections has prompted an urgent need for innovative alternatives to conventional antibiotics. Bacteriophages (phages) have emerged as a potential treatment option as naturally occurring viruses capable of killing bacteria. Inhaled delivery of phages offers targeted and localized action against the bacteria at the lung infection site. Phages are extremely sensitive and may lose viability during manufacturing and inhaled delivery (1). Post-deposition phages can undergo non-specific interactions with the lung epithelial cells. Consequently, phages available for clearing extracellular bacteria Pseudomonas aeruginosa could be greatly reduced (2). To address these challenges, this study investigates the encapsulation of phages in liposomes for phage stabilization during inhalation. The objective is to understand: (i) factors influencing liposomal phage encapsulation, (ii) the effect on phage aerosolization, and (iii) phage cellular uptake in a lung epithelial cell model.

Methods: In this study, bacteriophage (ATCC 14211-B1) targeting bacteria P. aeruginosa (ATCC 14211) was propagated and quantified using the plaque assay. The phage titer was measured in terms of plaque-forming units (PFU). Phage liposomes were prepared by the thin film hydration method, consisting of lipids HSPC, DSPG-Na, DSPE, and cholesterol (3:2:0.5:1.7 mass ratio) (3). Two phage loading titers were tested - 1.98 × 10⁷ and 5.49 × 10⁸ PFU/mL. Phage liposomes were downsized using syringe filtration (0.44 µm and 0.2 µm) followed by extrusion (0.1 µm, at 40°C). Free phages were separated by size-exclusion chromatography using a Sephadex G-25 column. Encapsulated phages were quantified by lysis using sodium deoxycholate (0.01% v/v). Phage encapsulation was calculated as a percent relative ratio of encapsulated phages to total phages. Phage liposomes were characterized using dynamic light scattering, and release was studied in simulated lung fluid (SLF) at 37°C. To evaluate the effect of aerosolization and in vitro lung deposition, nebulization was performed using PARI Proneb Max Aerosol system with LC plus nebulizer. Phage viability was studied using a filter-valve system to recover the nebulized phage. Phage cellular uptake was tested in an in vitro lung epithelial cell model NCI-H441 cells (500,000 cells/cm²) in a transwell with an area 0.33 cm²) grown at an air-liquid interface. Briefly, phage liposomes/suspension (2.82 × 10⁶ PFU/mL , 0.2 mL) were added to the apical chamber and HBSS to the basal chamber (0.6 mL), followed by 4-hour incubation. Samples were collected from both chambers, cell monolayers were lysed, and phage titer was determined using plaque assay. The distribution of labeled phages (SYBR-Gold) in the cell monolayer was studied using confocal microscopy.

Results: Phage liposomes prepared using two different hydration titers 1.98 × 10⁷ and 5.49 × 10⁸ PFU/mL, yielded low-titer (LT) and high-titer (HT) liposomes respectively. Processing conditions such as temperature and shear stress impact phage viability (5). The HT liposomes demonstrated a significantly higher loss in phage titer (p < 0.0001), compared to the loading titer. While no significant loss was observed in LT liposomes (Figure 1A). Two reasons contribute to phage loss: (1) loss due to processing conditions, and (2) separation of unencapsulated phages. Figure 1B, demonstrates that HT liposomes show significant loss (p < 0.01) of free phages on SEC compared to LT liposomes. Despite the observed loss, both HT and LT liposomes show similar encapsulated phage titer 6.59 x 10⁶ PFU/mL and LT 3.66 x 10⁶ PFU/mL, respectively. Conclusively, LT liposomes show higher phage encapsulation (58.33 ± 6.02 %) than HT (25.15 ± 5.67 %), hence LT liposomes were considered for further experimentation. The release profile (Figure 2A) of phage liposomes in SLF show small burst release of 10%, and slow controlled release of 35.15 ± 9.50 % up to 8 hours. Between 8-10 hours, all the encapsulated phages (98.13 ± 13.29 %) were released. The effect of aerosolization was studied using a jet nebulizer (Figure 2B). Phage liposomes demonstrated a significantly lower loss (1.08 ± 0.05 log) in titer as compared to free phages (1.55 ± 0.04). Phage cellular uptake was studied in a lung epithelial cell monolayer, grown at air-liquid interface. After 4-hour incubation, more phage liposomes (2.59 ± 0.30 × 10⁶ PFU/mL) were retained outside the cells as compared to free phages (2.10 ± 0.3 × 10⁶ PFU/mL) (Figure 3A). Consequently, the cell lysates of phage liposomes (1.42 ± 0.16 × 10⁴ PFU/mL) had significantly lower titer than free phage (2.91 ± 0.28 × 104 PFU/mL) (Figure 3B). This observation suggests that encapsulation of phages in liposomes can reduce their cellular uptake by 2-fold. This effect was also observed in confocal imaging (Figure 3C).

Conclusion: Our results demonstrate the potential utility of liposomes for inhaled phage delivery. Encapsulation of phage liposomes controls the release of phages, minimizes the loss due to aerosolization, and reduces the internalization in epithelial cells. All these advantages potentiate phages available for clearing respiratory infections.

References: 1. Abedon ST: Phage therapy of pulmonary infections. Bacteriophage 2015, 5: e1020260.
2. Bichet MC, Chin WH, Richards W et al.: Bacteriophage uptake by mammalian cell layers represents a potential sink that may impact phage therapy. iScience 2021, 24. doi: 10.1016/j.isci.2021.102287.
3. Yu S, Yuan H, Chai G et al.: Optimization of inhalable liposomal powder formulations and evaluation of their in vitro drug delivery behavior in Calu-3 human lung epithelial cells. International Journal of Pharmaceutics 2020,586: 119570.
4. Salomon JJ, Muchitsch VE, Gausterer JC et al.: The Cell Line NCI-H441 is a useful in vitro model for transport studies of human distal lung epithelial barrier. Mol Pharmaceutics 2014, 11: 995–1006.
5. Loh B, Gondil VS, Manohar P et al.: Encapsulation and delivery of therapeutic phages. Applied and Environmental Microbiology 2021, 87: e01979–20.

Acknowledgements: This work is supported by the National Institutes of Health under Award Numbers of R01AI146160 and R01HL167828. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


(A) Phage viability (in terms of phage titer) measured at different stages in the preparation of liposomes. Phage titer at each stage is compared to the initial loading titer (Tukey’s multiple comparison test, ****p < 0.0001) (B) Overall log reduction in phage titer observed before and after SEC (paired t-test, **p < 0.01) and among the formulations (unpaired t-test) (**p < 0.01, ***p < 0.001).


(A) Release pattern of phages from liposomes in simulated lung fluid at 37°C, (B) Effect of nebulization on phage titer using air-jet nebulizer PARI LC-Plus


The distribution of phages (suspension/liposomes) when incubated with H441 cell monolayer at air-liquid interface for 4 hours. Phage titer in A) apical chamber, B) cell lysates, C) Confocal images of phage cellular uptake in H441 cell monolayer