This study focuses on the development of a micron-sized, drug delivery vehicle - containing a superheated PFC phase - which releases the drug payload upon ADV of the PFC. Other groups formulating drug-carrying PFC emulsions have focused on submicron sized droplets (Fang et al. 2007
; Hwang et al. 2009
; Rapoport et al. 2007
). The use of micron-sized droplets that are transpulmonary enables the coupling of ADV-induced drug delivery and localized occlusion. Localized occlusion results from relatively large microbubbles blocking perfusion at the capillary level. This selective generation of transient, vascular occlusion has been previously demonstrated in vivo
(Kripfgans et al. 2002
). ADV-induced occlusion can complement ADV-induced drug delivery in two ways. First, the ischemic conditions created could increase the residence time of therapeutic material at the intended site, resulting in greater diffusion into the target tissue. A simple example of this phenomenon is obtained by comparing the 50%GI concentration for CHO cells when incubated with CHL for either 15 minutes (167 μM) or 60 minutes (57 μM). Second, the ischemic conditions can generate hypoxia that could be used to activate bioreductive prodrugs (McKeown et al. 2007
) that may be encapsulated within the emulsion. Thus, the proof-of-concept studies presented here serve as a foundation in developing future therapies that incorporate ADV.
The emulsion discussed in this work was prepared using a single emulsification step with only an aqueous-soluble surfactant, similar to other drug-carrying PFC emulsions (Fang et al. 2007
) and contrast agents (Unger et al. 1998
; Tartis et al. 2006
; Eisenbrey et al. 2009
). In contrast, double emulsions are typically prepared in two stages with two types of surfactants, where the innermost droplets are first emulsified followed by their subsequent emulsification in a secondary fluid (Goubault et al. 2001
). The presence of natural emulsifiers within soybean oil, including phospholipids (Sonntag 1988
) may help stabilize the PFP core.
The effect on the ADV threshold of encapsulating the PFP core within a layer of oil is currently unknown, though from it does not appear to be as significant for smaller droplets as larger droplets. In the case of AALs, the presence of an oil layer causes the pulse length required for contrast agent destruction to increase at least five-fold relative to contrast agent without an oil shell (May et al. 2002
). The thickness of the oil-lipid layer in AALs is 500 to 1000 nm and 300 to 700 nm for AALs containing triacetin and soybean oil, respectively (May et al. 2002
). By comparison, the mean oil-albumin layer thickness for the dual-phase droplets described in this study, measured using optical microscopy, is 1390±720 nm. It is possible that the oil layer could inhibit or dampen the expansion of any gas nuclei generated within the PFP core during the ADV process, possibly even causing a recondensation of the PFP at lower rarefactional pressures. This is similar to results where the ADV threshold of PFP droplets increased as the viscosity of the bulk fluid containing the droplets increased (Fabiilli et al. 2009
Concerning the %GI observed for group 2, PFC emulsions are known to cause cellular growth inhibition, but only in phagocytic cell lines (Bucala et al. 1983
; Centis et al. 2007
). While this %GI was well explained by droplet-cell adhesion and subsequent detachment from the OptiCell™ during washing, it was also hypothesized that the %GI experienced by group 2 might be attributable to the fact that PFC emulsions can modulate the oxygen content of media due to their high gas dissolving capabilities (Lowe et al. 1998
; Riess 2001
). Based on the amount of PFP injected into each chamber and the solubility of oxygen in PFP – 80% (v/v) (Johnson et al. 2009
) - the maximum change in the oxygen concentration within the chamber, assuming that the PFP did not initially contain any oxygen, is 10%, by weight. Hypoxic conditions can decrease the growth rate of CHO cells, but only when the cells are exposed to an oxygen concentration less than 3.5%, relative to the atmospheric oxygen concentration, for prolonged periods (i.e. > 10 hrs) (Lin and Miller 1992
). By comparison, after injecting the emulsion into the OptiCell™, the measured change in dissolved oxygen concentration was negligible (i.e. less than 1%) over one hour. This is likely due to the gas permeable nature of the OptiCell™ windows and that the PFP was partially saturated with oxygen prior to injection due to atmospheric contact.
The cellular bioeffects of ADV are currently unknown, though some insights can be obtained from the presented studies. The cell detachment due to the ADV process may be a result of droplet displacement prior to vaporization, where velocities up to 20 m/s have been recorded (Kripfgans et al. 2004
). Additionally, the rapid consumption of the liquid PFP and expansion of the resulting bubble during the ADV process (Haworth and Kripfgans 2008
), combined with bioeffects stemming from cavitation (Dalecki 2004
), could cause cell detachment. It is unknown whether ADV could cause cell detachment in vivo
or if the vaporization process could cause increased cell or vascular permeability, similar to results observed with microbubble cavitation (Hernot and Klibanov 2008; Pua and Zhong 2009
Since the intended mechanism of drug release from the dual-phase emulsion is US, the ADV efficiency is directly related to the amount of oil, and hence CHL, that could be potentially available for cellular exposure. As seen in , an inverse trend exists between the ADV threshold and the droplet diameter. Though this trend is confounded by the use of polydisperse droplets, it is hypothesized that droplets with a lower ADV threshold will also display a higher ADV efficiency for a given acoustic exposure. The inverse relationship between ADV threshold and droplet diameter is similar to the relationship between the thermal vaporization temperature of PFP droplets and droplet diameter. Using the Laplace pressure and Antoine equations, the diameter above which PFP droplets will undergo thermal vaporization at 37°C is 6.4 μm and 4.0 μm for surface tensions of 50 mN/m and 30 mN/m, respectively (Rapoport et al. 2009
); shell effects beyond surface tension reduction are ignored in these estimates. Therefore, the distinct ADV thresholds and defined ADV efficiencies of the emulsions, coupled with the observation that PFP emulsions are thermally stable up to 60°C (Kripfgans et al. 2000
), indic ate these emulsions are relatively free of nuclei that enable the emulsion to stably exist in a superheated state.
lists the fraction of total droplets vaporized in terms of both number and volume weighted distributions. The droplets in the 1 to 6 μm range are transpulmonary, and thus amenable to intravenous administration, whereas the droplets in the 6 to 30 μm range are amenable to intra-arterial administration. The number and volume-weighted fractions (without volume correction) were estimated by using the respective distributions and ADV efficiency in . The volume corrected fractions were estimated by correlating the ratio of the inner to outer diameter to the outer droplet diameter (). This correction was then applied to determine the volume of oil, and thus CHL, released upon ADV. The correction has a more significant impact on the larger droplets that, as seen in , have a larger PFP core. Thus, for a single pass of the US array – which was used in the cell experiments – approximately 50% of the oil is released upon ADV. Assuming an equal distribution of CHL within the oil, the actual concentration of CHL released via ADV in the chamber is 50 μM, which causes a 51%GI (based on ). In , group 8 displayed an 84.3%GI, which when the %GI caused by the emulsion alone (groups 2 and 6) is subtracted, the %GI from released CHL is approximately 64.3%. This is qualitatively consistent with the %GI predicted by the ADV efficiency.
Table 3 The fraction of total droplets vaporized, expressed in terms of number and volume fraction. The data without volume correction is the fraction of droplets vaporized, based on . The volume corrected data uses the relationship between the inner (more ...)
In summary, a stable, superheated, micron-sized emulsion has been developed that carries a lipophilic, therapeutic agent. The emulsion is triggered via US to produce gas bubbles and enhance the release of the encapsulated agent, as demonstrated with cultured cells. Current efforts are focused on increasing the drug loading of the dual-phase emulsions, minimizing the effect of non-US related drug release, and controlling the size distribution of the emulsions.