Preserving enhancement in freeze-dried contrast agent ST68: Examination of excipients

Abstract

The perfluorcarbon (perfluorobutane) ultrasound contrast agent ST68, composed of sonicated mixtures of non-ionic surfactants, is stable in solution for only a few weeks at 4°C. Freeze-drying critically diminished ST68’s ability to reflect ultrasound (its echogenicity). A method of incorporating specific lyoprotectants before lyophilization was investigated. Reintroduction of perfluorobutane to the protected freeze-dried sample, followed by reconstituting with preserved echogenicity. Glucose, trehalose, sucrose, and mannitol were tested at 100 mM and in vitro echogenicity data was collected from samples with dose concentrations of 50 µl/l to 300 µl/l. Glucose was found to be the best lyoprotectant providing an average (n=3) maximum peak enhancement of 23.2 ± 1.2 dB in vitro, measured at 5 MHz, 684 kPa, and a pulse repetition frequency (PRF) of 100 Hz (p<0.05 over freeze-dried ST68 control) and 20.8 ± 0.8 dB in vivo in New Zealand white rabbits at 5 MHz and a PRF of 6.7 kHz. Pulse inversion harmonic US images of a rabbit kidney, pre- and post-contrast injection (0.1 ml/kg), showed excellent enhancement and clear vascular delineation, similar to that of the original agent. For the first time this contrast agent can be successfully freeze-dried yielding a longer self-life without the need for refrigeration.

Development of an ultrasound sensitive oxygen carrier for oxygen delivery to hypoxic tissue

“…2 left), showed no ultrasound enhancement, indicating a lack of gas within the vials when capped under vacuum. Thus, we have ruled out the presence of residual octafluoropropane (which shows considerable enhancement (Solis et al, 2010)) acting as a trapped species, and hypothesize that the surfactant-based shell provides a superior resistance to gas diffusion across the microbubble wall.…”

“…The 2.5 ml samples in lyophilization vials were freeze-dried as described in the literature (Solis et al, 2010). In brief, Fluortec© lyophilization stoppers were inserted into the vials to the first groove (leaving a gap for air to escape).…”

“…The low toxicity of microbubbles, combined with the ability to control microbubble size, provides a safe and easily customizable platform for delivery of bioactive gases (Forsberg et al, 2010; Wheatley et al, 2006). Finally, a recent method of freeze-drying these particles under vacuum (Solis et al, 2010) allows the nascent bubble to be preserved for extended periods of time and resuspended with a desired gas. In this manuscript we characterize the ability of a microbubble agent to noninvasively elevate O 2 concentrations as a mechanism for overcoming tumor hypoxia-associated radiotherapy resistance.…”

Freeze-Dried Therapeutic Microbubbles: Stability and Gas Exchange

“…After refilling the vial with the appropriate gas, these cavities are filled and MBs are released upon rehydration. This has been used by Wheatley’s group in surfactant-based contrast agents (such as SE61, composed of Span60 and water-soluble vitamin E) to change the octa-fluoropropane gas core to O 2 to increase the O 2 level in hypoxic tissue upon delivery and, by the same group, to preserve the integrity of contrast agents with the non-ionic ST68 . The freeze-drying process has also been employed to increase the shelf life of polymeric MBs.…”

Pro-apoptotic liposomes-nanobubble conjugate synergistic with paclitaxel: a platform for ultrasound responsive image-guided drug delivery

“…The US-responsive platforms emerged from the serendipitous discovery of microbubbles (MBs) by Gramiak and Shaw in 1968 3 . Since then MBs have covered long and successful journey to clinics as ultrasound contrast agents (UCAs).…”