The surface charge of liposomal adjuvants is decisive for their interactions with the Calu-3 and A549 airway epithelial cell culture models

• We tested the effect of liposomal adjuvant charge on interaction with lung cells.
• Cationic liposomes deposited most efficiently lipid and antigen in the cells.
• The cationic liposome charge is important for the delivery.
• Mucus retained the integrity/viability upon incubation with cationic formulations.
• Cationic liposomes might be safely used for mucus-covered epithelium.

One of the main reasons for the unmet medical need for mucosal vaccines is the lack of safe and efficacious mucosal adjuvants. The cationic liposome-based adjuvant system composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6′-dibehenate (TDB) is a versatile adjuvant that has shown potential for mucosal vaccination via the airways. The purpose of this study was to investigate the importance of the liposomal surface charge on the interaction with lung epithelial cells. Thus, the cationic DDA in the liposomes was subjected to a step-wise replacement with the zwitterionic distearoylphosphatidylcholine (DSPC). The liposomes were tested with the model protein antigen ovalbumin for the mucosal deposition, the effect on cellular viability and the epithelial integrity by using the two cell lines A549 and Calu-3, representing cells from the alveolar and the bronchiolar epithelium, respectively. The Calu-3 cells were cultured under different conditions, resulting in epithelia with a low and a high mucus secretion, respectively. A significantly larger amount of lipid and ovalbumin was deposited in the epithelial cell layer and in the mucus after incubation with the cationic liposomes, as compared to incubation with the neutral liposomes, which suggests that the cationic charge is important for the delivery. The integrity and the viability of the cells without a surface-lining mucus layer were decreased upon incubation with the cationic formulations, whereas the mucus appeared to retain the integrity and viability of the mucus-covered Calu-3 cells. Our in vitro results thus indicate that DDA/TDB liposomes might be efficiently and safely used as an adjuvant system for vaccines targeting the mucus-covered epithelium of the upper respiratory tract and the conducting airways.

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