The use of
injectable gas-filled
microbubbles during ultrasound imaging is accepted as a good method to increase image contrast. Site-targeted
microbubbles are expected to provide higher sensitivity and specificity than blood pool
contrast agents (CAs). We have shown that covalent attachment of
GRGDS peptide fragments to the surface of
poly(lactic acid) CAs facilitates attachment to MDA-MB-231 human
breast cancer cells in vitro. This paper examines the effect of process conditions during
microbubble fabrication and
ligand attachment and also changes in
ligand surface density and shows that they have important effects on in vitro acoustic response and CA adhesion to
breast cancer and cell lines. Use of intermittent sonication in the
emulsion step, shortening of reaction times, and increase in freeze-drying times allows for a reduction of 50% in the dose of
GRGDS-modified capsules (from 0.16 to 0.012 mg/mL) required to achieve a maximum enhancement of 20 dB; signal loss after 15 min insonation of
GRGDS-modified capsules is reduced from a loss of 60% to a loss of 40%, and cell attachment after 10 min contact time is increased from an average of 1.4 +/- 0.86 to 1.8 +/- 0.17 capsules/cell. Optimal attachment is achieved with a molar ratio of total -COOH groups to
GRGDS of 1:0.5. The effect of process conditions during
microcapsule fabrication,
ligand attachment, and
ligand surface density on in vitro acoustic response and CA adhesion to
breast cancer cell lines in tissue culture are shown to be important parameters that can aid in the future design of an ultrasound CA that allows both
cancer detection and treatment, potentially by targeted
drug delivery.