Respiratory tract infections induced by antimicrobial-resistant (AMR) bacterial pathogens due to global antibiotic abuse have been considered as a leading cause of suffering and death worldwide (Uddin et al., 2021), where Staphylococcus aureus represents a major fatal agent (Tong et al., 2015). In addition to the AMR crisis, the unique ability of S. aureus to invade pulmonary epithelial/endothelial cells and cause intracellular infections has become a tricky clinical issue (Strobel et al., 2016). The intracellular invasion can allow the pathogens to evade the host immune clearance and invalidate antibiotics that have limited cellular internalization, making lung infections difficult to treat and usually recurrent (Fisher et al., 2017). The disappointing outcomes emphasize the urgent necessity to explore approaches against the intracellularly-invasive S. aureus.

In recent years, using lytic (virulent) bacteriophage (phage) to specifically target and kill bacteria has been regarded as a promising alternative to antibiotics in targeting AMR bacterial infections (Yang et al., 2021). Promisingly, phages are produced as an emergency investigational new drug (eIND) for compassionate use under US Food and Drug Administration (USFDA) approval and its efficacy has been validated in several clinical cases/trials (Dedrick et al., 2019, Jault et al., 2019, Schooley et al., 2017). Even so, the effectiveness of phages handling intracellular bacterial infections is rarely reported and phages are commonly known not to interact with eukaryotic cells (Tetz et al., 2017). Although some recent literature revealed that phages could be internalized into mammalian cells, depending on the cell types, phage size and morphology (Bichet et al., 2021), the sufficient exposure of phages inside mammalian cells to eliminate the resided bacteria is questionable. Therefore, development of suitable formulations to enhance phage intracellular delivery is warranted.

As the first and only approved nanocarrier for lung delivery to date, liposomes are expected to enhance the intracellular drug delivery by virtue of the biocompatibility of phospholipid bilayers to the cell membrane (Bulbake et al., 2017, Zheng et al., 2022). Liposomal-phage has attracted increasing attention in the academia with satisfactory progress (Wang et al., 2021). Nieth et al. reported the liposome-associated phages were able to enter THP-1 cultured eukaryotic cells, more efficiently than free phages (Nieth et al., 2015). Work by Vladimirsky et al. on the Mycobacterium tuberculosis-infected macrophage cell culture (RAW 264–7) showed that liposomal mycobacteriophage had significantly higher bactericidal effect than the free phage (Mikhail et al., 2019). These studies highlighted the potential role of liposomes in the delivery of phage to target intracellular pathogens. Employing liposomal phage to treat respiratory bacterial infections, pulmonary delivery will be a promising strategy. To the best of our knowledge, the aerodynamic performance of liposomal phage formulations and their stability during aerosolization has not yet been investigated. It has been reported that the nebulization capability and aerodynamic deposition of inhaled formulations are associated with the composition of lipid vesicles (Altube et al., 2023). Furthermore, drug loss/inactivation and particle aggregation, which are commonly seen in the nebulized aerosols, are also related to the lipid type comprised (Szabová et al., 2021). In other words, nebulization stability remains a challenge for liposomal drugs to overcome, which is closely linked to their further clinical use and efficacy.

To fill in these knowledge gaps, we developed in this study a novel, self-assembling liposome-phage nanocomplex formulation, focusing on its nebulization performance together with favorable therapeutic effects against intracellular infections. In this context, we selected three commonly used phospholipids, including soybean phosphatidylcholine (SPC), egg phosphatidylcholine (EPC), and hydrogenated soybean phosphatidylcholine (HSPC), to form liposomes with cholesterol, followed by formation of nanocomplexes with phage K. The ability of these liposomes in protecting phage K against nebulization-induced stress, depositing in lower respiratory tract, and killing intracellular S. aureus were investigated. Our results indicated that the lipid type played an important role in promoting the nebulization efficiency and intracellular bacterial targeting of phage K. This study demonstrated a promising therapeutic strategy for treating intracellular infections and proposed an efficient inhalable phage formulation for pulmonary delivery.