Development of Inhalable Bacteriophage Liposomes Against Pseudomonas aeruginosa

Abstract

Background:Pseudomonas aeruginosa is one of the major pathogens that cause respiratory infections. The rise of antimicrobial resistance has prompted a need for alternatives to conventional antibiotics. Bacteriophages (phages), natural predators of bacteria, are gaining interest as an alternative therapeutic option against drug-resistant infections. However, phage viability can be lost during manufacturing and delivery. Recent studies show that phages can be taken up by lung epithelial cells, which makes fewer phages available for antibacterial action against extracellular bacteria P. aeruginosa in the airways. Methods: In this study, we encapsulated phages in liposomes using thin film hydration. The effect of processing conditions and phage loading titer on the phage encapsulation and viability was studied. The impact of nebulization on phage viability was tested using an air-jet nebulizer (PARI-LC Plus). Phage cellular uptake was evaluated using an in vitro H441 lung epithelial cell model, grown at the air-liquid interface. Results: Our results demonstrate favorable encapsulation (58 ± 6.02%) can be achieved with minimum loss in phage titer (0.64 ± 0.21 log) by using a low phage titer for hydration. The liposomal formulations exhibited controlled release of phages over 10 h. The formulation also reduced the loss of phage viability during nebulization from 1.55 ± 0.04 log (for phage suspension) to 1.08 ± 0.05 log (for phage liposomes). Encapsulation of phages in liposomes enabled a two-fold reduction in phage cellular uptake and longer extracellular phage retention in the human lung epithelial cell monolayer. Conclusions: Our results indicate that liposomal encapsulation favors phage protection and improves phage availability for antibacterial activity. These findings highlight the potential of liposomes for inhaled phage delivery.

Keywords: bacteria; inhalation; liposomes; nebulization; phages; pulmonary delivery.

Figure 1

(A) Phage viability (in terms of phage titer) measured at different stages in the preparation of liposomes. The phage titer at each stage was compared to the initial loading titer (Tukey’s multiple comparison test, **** p < 0.0001, ns = no significant difference). (B) Overall log reduction in phage titer observed before and after SEC (paired t-test, ** p < 0.01) among the formulations (unpaired t-test) (** p < 0.01, *** p < 0.001, ns = no significant difference). Each data point represents mean ± SD, (n = 3).

Figure 2

(A) Release patterns of phages from liposomes in simulated lung fluid at 37 °C. (B) Phage titer of released phages and total viable (encapsulated + released) phages present at different time points (hours) in the release study. Each data point represents mean ± SD, (n = 3).

Figure 3

Phage deposition profiles across different stages of the MSLI following nebulization with PARI LC Plus nebulizer. Percentage deposition is relative to the phages recovered after aerosolization through the nebulizer. Each data point represents mean ± SD, (n = 3).

Figure 4

The distribution of phages (suspensions/liposomes) when incubated with H441 cell monolayer at air–liquid interface for 4-h (n = 4). Phage titer in (A) apical chamber, (B) cell lysates, and (C) percentage phage recovered after 4 h. Data represent four replicates, with treatment groups compared using an unpaired t-test (** p < 0.01, and **** p < 0.0001).

Figure 5

Representative confocal images of H441 cell monolayers after 4-h treatment with phage suspension or liposomes. SYBR Gold-labeled phages in green, Hoechst 33342 stained nuclei in blue, and rhodamine-labeled liposomes in red.