Phosphatidylcholine-coated microbubbles (PC-MBs) are used clinically in contrast-enhanced ultrasound (CEUS) for the characterization of solid tumor perfusion¹. CEUS uses the nonlinear echoes of MBs to differentiate them from solid tissues. This study demonstrates that PC-MBs exhibit increased nonlinear echoes in response to changes in pH, detectable by clinical ultrasound scanners. Interestingly, this phenomenon occurs in the pH range found in acidotic solid tumors. By harnessing the response of PC-MBs to acidosis, CEUS techniques may be developed to assess intratumoral pH. Given the fact that tumor microenvironment acidosis is a determinant of the local aggressiveness and metastatic potential of solid tumors^2,3, these techniques would provide insight into the prognosis of solid tumors and their response to therapy, with the ultimate goal of guiding individualized treatment³.

MBs composed of a perfluorobutane gas core and a shell of either PC (90% carbon chain lengths of 14 to 20 atoms; 10% PEGylated lipid) or distearoyltrimethylammoniumpropane (DSTAP) were added to PBS (37°C) within a flexible pipet bulb, and imaged inside a tissue-mimicking phantom⁴. PBS pH was adjusted using aqueous NaOH or HCl. MBs were imaged by a clinical ultrasound scanner at 7.0 MHz and 1.0 MHz in both B-mode and Cadence pulse sequencing (CPS) mode. MB brightness was analyzed by ImageJ. Lumason (Bracco Diagnostics), a PC-MB formulation used clinically, was tested in a similar fashion.

A 2- to 3-fold increase in CPS signal intensity of PC-MBs was observed in response to a pH decrease from 7.4 to 6.0, at both 7.0 and 1.0 MHz. Interestingly, this change in signal intensity was reversible following increase to the initial pH. Lumason exhibited a less robust increase in signal intensity, which may be due to shell composition (only 50% PC). Examination of activation kinetics of PC-MBs with different carbon chain length PCs demonstrated that shorter carbon chain lengths exhibited more rapid activation when pH was decreased. Previous studies of PC monolayers at the air-water interface demonstrate decreased surface tension in a similar pH range, providing as explanation for this behavior of PC-MBs⁵. This property was attributed to changes in molecular packing of PCs. Stronger intermolecular forces between longer carbon chains may therefore require greater time to exhibit change in conformation⁶. Unlike PC-MBs, MBs composed of DSTAP (which contains no phosphate) exhibit no acid-activation. Therefore, despite the low pKa of phosphate in PCs⁷, hydronium interaction with phosphate groups may be required to cause changes in monolayer conformation. We hypothesize that this interaction results in a more cohesive lipid monolayer, causing the shell to resist fracture and change shell elasticity, resulting in increased CPS signal intensity^6,8.

While harmonic signal of solid tumors in CEUS studies is typically attributed to blood flow, it is possible that local pH also impacts signal intensity. Furthermore, these findings suggest that this effect can be quantified, potentially allowing the measurement of deep tissue pH noninvasively. Taken together, these findings could pave the way for the development of novel bioresponsive ultrasound contrast agents.

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Image/Figure Caption:

Phosphatidylcholine-coated microbubbles exhibit marked increased nonlinear oscillation when ambient pH is decreased from 7.4 to 6.5, resulting in marked increase in signal intensity when imaged using Cadence contrast pulse sequencing (CPS, a technique used to measure nonlinear echoes).

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