Penetration of biofilm and targeted, sustained release from liposomes can explain the superior in vivo efficacy of inhaled liposomal amikacin versus free drug observed in a 14 day infection model. Inhaled liposomal amikacin may represent an important therapy for chronic lung infections.

We reported previously the effects of both osmotic and curvature stress on fusion between poly(ethylene glycol)-aggregated vesicles. In this article, we analyze the energetics of fusion of vesicles of different curvature, paying particular attention to the effects of osmotic stress on small, highly curved vesicles of 26 nm diameter, composed of lipids with negative intrinsic curvature. Our calculations show that high positive curvature of the outer monolayer "charges" these vesicles with excess bending energy, which then releases during stalk expansion (increase of the stalk radius, r(s)) and thus "drives" fusion. Calculations based on the known mechanical properties of lipid assemblies suggest that the free energy of "void" formation as well as membrane-bending free energy dominate the evolution of a stalk to an extended transmembrane contact. The free-energy profile of stalk expansion (free energy versus r(s)) clearly shows the presence of two metastable intermediates (intermediate 1 at r(s) approximately 0 - 1.0 nm and intermediate 2 at r(s) approximately 2.5 - 3.0 nm). Applying osmotic gradients of +/-5 atm, when assuming a fixed trans-bilayer lipid mass distribution, did not significantly change the free-energy profile. However, inclusion in the model of an additional degree of freedom, the ability of lipids to move into and out of the "void", made the free-energy profile strongly dependent on the osmotic gradient. Vesicle expansion increased the energy barrier between intermediates by approximately 4 kT and the absolute value of the barrier by approximately 7 kT, whereas compression decreased it by nearly the same extent. Since these calculations, which are based on the stalk hypothesis, correctly predict the effects of both membrane curvature and osmotic stress, they support the stalk hypothesis for the mechanism of membrane fusion and suggest that both forms of stress alter the final stages, rather than the initial step, of the fusion process, as previously suggested.

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Poly (ethylene glycol) (PEG) in the external environment of membrane vesicles creates osmotic imbalance that leads to mechanical stress in membranes and may induce local membrane curvature. To determine the relative importance of membrane stress and curvature in promoting fusion, we monitored contents mixing (CM) and lipid mixing (LM) between different sized vesicles under a variety of osmotic conditions. CM between highly curved vesicles (SUV, 26 nm diameter) was up to 10 times greater than between less curved vesicles (LUV, 120 nm diameter) after 5 min incubation at a low PEG concentration (<10 wt%), whereas LM was only approximately 30% higher. Cryo-electron microscopy showed that PEG at 10 wt% did not create high curvature contacts between membranes in LUV aggregates. A negative osmotic gradient (-300 mOs/kg, hypotonic inside) increased CM two- to threefold for both types of vesicles, but did not affect LM. A positive gradient (+220 mOs/kg, hypertonic inside) nearly eliminated CM and had no effect on LM. Hexadecane added to vesicles had no effect on LM but enhanced CM and reduced the inhibitory effect on CM of a positive osmotic gradient, but had little influence on results obtained under a negative osmotic gradient. We conclude that the ability of closely juxtaposed bilayers to form an initial intermediate ("stalk") as soon as they come into close contact was not influenced by osmotic stress or membrane curvature, although pore formation was critically dependent on these stresses. The results also suggest that hexadecane affects the same part of the fusion process as osmotic stress. We interpret this result to suggest that both a negative osmotic gradient and hexadecane reduce the unfavorable free energy of hydrophobic interstices associated with the intermediates of the fusion process.

The stress of nebulization has been shown to alter the properties of liposomal drugs. What has not been demonstrated is whether nebulized liposomes differ as a function of droplet size. Because droplet size influences lung deposition, liposomes with different properties could be deposited in different areas of the lung (e.g., central vs. peripheral). In this report, a liposomal amikacin formulation (Arikace, a registered trademark of Transave, Inc., Monmouth Junction, NJ) that is being developed as an inhaled treatment for gram negative infections was aerosolized with an eFlow (registered trademark of PARI, GmbH, Munich, Germany) nebulizer, reclaimed from the various stages of an Andersen cascade impactor (ACI) and analyzed for lipid-to-drug (L/D) (w/w) ratio, amikacin retention, and liposome size. For the nebulized solution, 99.7% of the total deposited drug was found on ACI stages 0 through 5, which have cutoff diameters of 9, 5.8, 4.7, 3.3, 2.1, and 1.1 microm, respectively. Properties were found to differ for drug reclaimed on stage 0 compared stages 1-5, which were not different from one another. For drug found on stages 1-5 (97% of total drug), the averages (n = 3) for L/D, percent encapsulated amikacin, and liposome mean diameter ranged from 0.59 to 0.68 (w/w), 71% to 75%, 248 to 282 nm, respectively. Drug found on stage 0 (2.8% of total drug) had an average L/D ratio of 0.51 and average liposome mean diameter of 375 nm. Examination of another batch of liposomal amikacin revealed no statistically significant differences between drug reclaimed on stages 0-5. Although a droplet size dependence was noted for one batch of Arikace aerosolized with the eFlow, the effect was considered to be inconsequential because the fraction in doubt represented nonrespirable particles >9 microm and accounted for <3% of the total deposited dose. The methodology applied here appears useful in evaluating aerosolized liposome systems. However, our results should not be assumed to apply to other liposome/drug compositions and nebulizers.