Pre-formulation studies on model microbubbles

However, the effect of MB degradation cannot be explained only by the temperature increase, since in follow-up studies MB dispersions, incubated for over 90 min at 60 °C under static conditions, showed only a moderate concentration decrease of about 17.5%. Apparently, the observed phenomenon can only be explained under consideration of the combined effect of temperature and shear stress.

A significant effect of the agitation time on the MB particle size distribution was also demonstrated, as shown on Figure 7. Initially, a single broad peak between 260 nm and about 10.1 µm was present in MB samples 20 s after agitation start. Longer agitation of 40 s resulted in a peak sharpening and narrowing to size values between about 800 nm and 5.8 µm.

Furthermore, a second small peak was formed between 34 µm and 77 µm. At 60 s agitation, the large-sized peak gained intensity and broadened between about 23 µm and 153 µm.

Figure 7: Surface-weighted MB particle size distribution as a function of the agitation time.

Microscopic observations revealed that during the first five to ten seconds of agitation the MB dispersion is rather “immature” and inhomogeneous, comprising micrometer- to millimeter-sized agglomerates of small and large bubbles and phospholipid (data not shown). Later on, after 20-30 s the phospholipid aggregates and large “primary” bubbles were sheared and a homogeneous MB dispersion was formed.

Obviously, the optimal MB size distribution was obtained 40 s after agitation start. However, this was accompanied by an about 1.5-fold decrease of MB concentration after 40 s agitation compared to 20 s. These results suggest that with regard to each particular drug-loaded

formulation, the optimal agitation time should be sought between 20 s and 40 s under consideration of the two factors – MB size distribution and yield.

4.1.2. Effect of liposome viscosity

Liposome viscosity increased almost linearly from 1.5 Pa.s to about 3 Pa.s upon increasing glycerol concentration. The influence of liposome viscosity on the production of MBs by mechanical agitation revealed complex trends both in the devolution of the tube temperature curve, as in the MB concentration.

By increasing the viscosity in the above range two maxima were observed in the tube temperature curve after 20 s of agitation (Figure 8, solid line). Initially, addition of 5 mass%

to 10 mass% glycerol led to a small but reproducible tube temperature increase of about 2 °C compared to glycerol-free samples. Interestingly, the moderate temperature increase was accompanied by an over 2-fold increase of MB concentration (Figure 8, dashed line).

Figure 8: Effect of liposome viscosity (at τ = 2 Pa) on the tube temperature and the MB yield at agitation time 20 s (n = 3). Dashed line represents the tube temperature after agitation for 20 s. Solid line represents the MB concentration in freshly prepared samples.

Further increase of viscosity resulted in an unexpected drop of tube heating of over 5 °C together with an insignificant decrease of MB concentration. Tube heating rate rose back to about 37 °C upon reaching a viscosity of 2.73 Pa.s. In general, a viscosity increase of 1.405 ± 0.054 Pa.s raised the MB yield about 10-fold. This was despite the enhanced tube

heating during agitation, since the gel-to-liquid phase transition of shell phospholipids – 41.55 °C ± 0.11 °C, had not yet been reached.

Apparently, glycerol had some more specific effects on the system rather than simply increasing the viscosity, since the non-linear character of both the temperature curve and the MB concentration curve did not correspond to the linearity of the viscosity increase at growing glycerol concentration.

Figure 9: Effect of viscosity (at τ = 2 Pa and 25 °C) on the MB mean size according to number-weighted particle size data.

Furthermore, viscosity had significant influence on the MB size distribution, yet this effect was only pronounced between 0 mol% and 5 mol% (Figure 9), in which range the viscosity increase was only 0.07 Pa.s. Thus, apparently this is also due to interactions between the phospholipids and glycerol, rather than an effect, caused by the viscosity.

Generally, addition of 5 mass% to 10 mass% of glycerol resulted in a sharpening and narrowing of the main MB peak between 500 nm and 7 µm. On surface area-weighted diagrams, the emerging of a second peak between 50 µm and 100 µm was observed at glycerol concentrations above 10 mass%.

4.1.3. Effect of tube fill volume

The proportion between the volume of liposomal dispersion and the total volume of of the tube container was denoted here as tube fill volume. It had a moderate, but significant influence on the MB yield (Figure 10), and a more pronounced effect on the MB size distribution. Generally, with increasing the tube fill volume from 10% to 50% of the total tube volume the MB concentration decreased approximately two times, as maximum yield was achieved at 20% tube fill volume.

Along with this, the fraction of large MBs in the size range of several hundred micrometers steeply increased. In surface area-weighted size diagrams only the MB peak between about 500 nm and about 6 µm was present at fill volumes of 10% and 20%. At 30% tube fill volume the size peak between about 60 µm and about 100 µm appeared, while further increase to 40%

and 50% of the tube volume resulted in the emerging of smearing peaks from about 200 µm to about 600 µm.

Figure 10: Effect of tube fill volume on the MB yield (m = 3, n = 3) after agitation for 20 s. Maximal MB yield was achieved, when 400 µl liquid phase – liposome precursor dispersion, were filled in the tubes, having a total volume of 2,000 µl (20% tube fill volume).

The observed effects can be explained with the downgraded mixing kinetics of gas and liquid, if the cap space is too small to allow adequate bouncing of the liquid phase during agitation.

At higher tube fill volumes of e.g. 50% a large part of the gas phase is absorbed into the liquid phase. Because of the MB formation, the dynamic viscosity of the liquid phase grows about

300-fold (data not shown). Therefore, the fluid dynamics in the tube are hindered and no sufficient shear forces can result.

This is an appropriate explanation for the reduced fraction of small MBs since at higher tube fill volumes more shell material is employed in larger bubbles.

4.1.4. Effect of tube shape

Tube geometry had a moderate effect on the MB formulation. No difference could be found between size distribution and concentration of MBs, produced in F-bottom and U-bottom tubes. When V-bottom tubes were used, there was a significant increase of MB size and a large-sized bubble peak between 60 µm and 120 µm emerged. Once again this effect could be attributed to the unadvantageous fluid dynamics during agitation, caused by the narrow V-shaped tube bottom.

4.2. Characterization of microbubbles