To address these limitations, researchers at the Sunnybrook Research Institute in Toronto, Canada, are developing slightly larger contrast agents that selectively extravasate from leaky vasculature. One prime candidate is perfluoro-octylbromide (PFOB), nanoscale droplets of which offers strong X-ray attenuation at CEDM energies and has been used previously in patients.

"An advantage of PFOB as a contrast agent is that it can be formed into droplets with a diameter of between 100 and 200 nm," explained researcher Melissa Hill. "The normal capillaries in the breast are impermeable to molecules of this size. This means that the PFOB droplets should only be able to extravasate from leaky tumour vasculature that has an abnormal capillary wall."

The team's ultimate goal is to engineer droplets with appropriate X-ray attenuation characteristics and size for use in CEDM. To evaluate the droplets in pre-clinical environments, and speed clinical translation, they labelled the droplets with fluorescent quantum dots (QDs) to enable independent validation of their spatial location (Phys. Med. Biol. 58 5215).

Fluorescent features

Hill and colleagues studied three materials: PFOB; QD-labelled PFOB; and a control perfluorocarbon (PFC) compound, perfluorotributylamine, which neither attenuates X-rays nor fluoresces.

They first measured the fluorescence emission spectra of the raw materials. When excited by 350 nm light, QD-PFOB exhibited a strong photoluminescence peak at 635 nm with negligible background, while pure PFOB and PFC exhibited little fluorescence. As all three materials exhibited negligible fluorescence at 585 nm, this wavelength was used as the background signal.

They then formed QD-PFOB droplets, using a biocompatible emulsifier to create a droplet-stabilizing shell. All droplets had similar mean sizes of about 160 nm (which was unaffected by adding QDs) making them suitable for intravascular tumour imaging. The strong QD-PFOB fluorescence was retained after droplet synthesis and purification, while PFOB and PFC droplets again exhibited only background fluorescence.

X-ray characterization

Next, the researchers performed X-ray characterization of the materials, using a custom-built table-top cone-beam CT system. They first measured the X-ray attenuation of raw PFOB at various concentrations in PFC.

PFOB strongly attenuated a 30.5 keV X-ray beam, with a relative attenuation of 5621±268 HU per g/ml PFOB. The authors note that the addition of QDs to the PFOB solution did not significantly affect the X-ray attenuation. Measurements on droplets of PFOB, QD-PFOB and PFC showed that the relative X-ray attenuations were retained after agent formation.

Tissue phantoms

Finally, Hill and colleagues studied the QD-PFOB droplets under more realistic in vivo conditions, using phantoms based on murine alveolar macrophage cells. Such phantoms provide autofluorescence and X-ray attenuation representative of normal tissue, against which the additional signal enhancement conferred by the PFOB and QD-PFOB droplets could be measured.

They loaded cells with QD-PFOB droplets mixed with PFC droplets at fractions of 0, 0.10, 0.25, 0.75, and 1.00. Fluorescence microscopy revealed that the QD-PFOB droplets were loaded into cells within the cell membrane and outside the nucleus.

To assess X-ray attenuation, the researchers centrifuged the cell suspensions into pellets and measured the change in attenuation due to the addition of QD-PFOB droplets. Increased loading of QD-PFOB droplets clearly increased the X-ray attenuation in comparison to pellets of unloaded control cells.

They also measured the dependence of the fluorescence on the concentration of QD-PFOB droplets, by suspending cells in saline and illuminating them with UV light. The fluorescent enhancement from cells loaded with a 0.10 QD-PFOB droplet mixture was clearly measurable, while PFC droplet-loaded cells were indistinguishable from unloaded cells.

The researchers note that the fluorescence enhancement of QD-PFOB in single cells directly correlated with the X-ray attenuation of the same cells in pelleted form. This strong correlation suggests that the QD-PFOB fluorescence can be used to evaluate the biological targeting potential of PFOB droplet formulations in vitro, at a cellular level, without requiring CT imaging.

"The next step is to move the agent in vivo, for optimization of the droplet formulation," Hill told medicalphysicsweb. "We are beginning by using a mouse tumour model for testing of droplet shell formulations that maximize tumour avidity and plasma half-life, while minimizing non-specific uptake."

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