The kinetics and mechanism of accumulation for liposomal ciprofloxacin (Lipo-CPFX) into the rat alveolar macrophage NR8383 cells were studied in vitro, in comparison to unformulated ciprofloxacin (CPFX). Upon incubation with CPFX or Lipo-CPFX, cellular drug accumulation was determined from the cell lysates or efflux was from the extracellular media by fluorescence-HPLC. The accumulation for Lipo-CPFX reached the asymptotic values at ≥ 2 hours, which was a result of uptake and efflux. The uptake appeared to be due to liposomes, mediated via cellular energy-independent mechanism like lipid fusion. In contrast, the efflux appeared to be due to ciprofloxacin, partly cellular energy-dependent, and involve probenecid-sensitive multidrug resistance proteins (MRPs). Overall, Lipo-CPFX enabled greater drug accumulation into the NR8383 cells than CPFX. This logically suggests a greater potential to treat respiratory infections especially caused by bacteria resistant to phagocytic killing.

Liposomal ciprofloxacin (Lipo-CPFX) is being developed for inhalation, with a goal of sustaining the therapeutic activity, compared to unformulated ciprofloxacin (CPFX). However, the kinetics and mechanism of its sustained local lung retention and pharmacological activity are yet to be fully characterized. This project hypothesized that Lipo-CPFX enables slower and sustained lung epithelial transport and uptake, compared to CPFX, thereby producing prolonged local pharmacological actions. The human bronchial epithelial Calu-3 cells were used as monolayers to characterize the kinetics and mechanism of transport and/or uptake, and to assess the effects of such slow kinetics for Lipo-CPFX on its inhibition against lipopolysaccharide (LPS)-induced proinflammatory IL-8 release. The transport fluxes for Lipo-CPFX across the highly restricted Calu-3 cell monolayers was transepithelial electrical resistance-independent, which suggested predominant transcellular transport. Compared to CPFX, Lipo-CPFX showed 6-18 times slower transport, while the flux was increased with increasing concentration proportionally without saturation. Its unaltered transport by cellular energy depletion, transport inhibition by a reduced temperature (4 oC) and endocytosis/lipid fusion inhibitors, filipin and LysoPC, and increased transport by excess empty liposomes collectively suggested cell energy-independent, lipid bilayer fusion mechanisms for the Lipo-CPFX transport across the Calu-3 cells. Likewise, Lipo-CPFX showed 2-4 fold lower cellular uptake than CPFX, proportional to concentration. Lipo-CPFX exhibited significant inhibitory activities at ≥ 0.01 mg/mL on LPS-induced IL-8 release from the Calu-3 cells, which was equipotent to CPFX. Upon 24 h pre-incubation, Lipo-CPFX caused 36.9 and 47.5 % inhibition at 0.01 and 0.05 mg/mL, respectively, while CPFX failed to do so. However, the effect was negated upon repeated wash of the mucosal cell surface, speculating the importance of cell membrane-associated drug/formulation on the inhibitory activities for Lipo-CPFX. Upon 24 h transport, Lipo-CPFX retained 79.0 % of the 4 µg dose on the mucosal cell surface, which was 1.9-times greater than 40.7 % for CPFX. As a result, when LPS was added at 24 h of the transport, Lipo-CPFX was still capable of causing 60.1 % inhibition, as its sustained local anti-inflammatory activity; CPFX however also exhibited equipotent inhibition, by virtue of comparable cellular drug uptake/transport.