Feasibility of lactadherin-bearing clinically available microbubbles as ultrasound contrast agent for angiogenesis

Phagocytosis of apoptotic cells is carried out through bridging of phosphatidylserine (PS)-expressing apoptotic cells and integrin αvβ3-expressing phagocytes with lactadherin. The objective of this study was to examine whether microbubbles targeted to integrin αvβ3 could be produced by conjugating a PS-containing clinically available ultrasound contrast agent with lactadherin.

PS-containing perfluorobutane-filled microbubbles were incubated with R-phycoerythrin (PE)-labeled lactadherin, and the presence of PE-positive bubbles was examined by FACS analysis. Secondly, the attachment of lactadherin to integrin αvβ3-expressing cells (human umbilical vein endothelial cells (HUVEC)) was also examined by FACS analysis. Finally, the adhesion of PS-containing bubbles to HUVEC was examined using a parallel plate flow chamber. The number of adherent bubbles with or without the intermediation of lactadherin was compared.

The more lactadherin was added to the bubble suspension, the more PE-positive bubbles were detected. The size of bubbles was not increased even after conjugation with lactadherin (2.90 ± 0.04 vs. 2.81 ± 0.02 μm). Binding between lactadherin and HUVEC was also confirmed by FACS analysis. The parallel plate flow chamber study revealed that the number of PS-containing bubbles adherent to HUVEC was increased about five times by the intermediation of lactadherin (12.1 ± 6.0 to 58.7 ± 33.1 bubbles).

Because integrin αvβ3 is well-known to play a key role in angiogenesis, the complex of PS-containing bubbles and lactadherin has feasibility as a clinically translatable targeted ultrasound contrast agent for angiogenesis.