Perfluoropentane-filled chitosan poly-acrylic acid nanobubbles with high stability for long-term ultrasound imaging in vivo

Abstract

Reducing the size of ultrasound contrast agents (UCAs) will decrease the intensity of the ultrasound echogenic signals and reduce the stability of the bubbles. Therefore, it is a challenge to design nanobubbles that are less than 200 nm in size and that have both good imaging abilities and high stability for long-term imaging in vivo. In this work, we successfully prepared perfluoropentane-filled chitosan poly-acrylic acid (PFP-CS-PAA) nanobubbles with a size of about 100 nm via a direct simple core-template-free strategy. In vitro tests demonstrated that the nanobubbles showed satisfactory imaging capabilities in non-linear harmonic imaging mode and had significantly better stability than commercial Sonovue® lipid microbubbles. It was valuable to discover that the prepared PFP-CS-PAA nanobubbles could exhibit good imaging quality in rat livers for 10 min after intravenous injection. Also, the PFP-CS-PAA nanobubbles could maintain imaging capabilities in nude mouse tumors for 7 days after intratumoral injection, which indicated that these nanobubbles could keep their stability for a long time in vivo. To the best of our knowledge, the ultrasound imaging persistence time in vivo was the longest of currently reported polymer nanobubbles that are smaller than 200 nm. This new nanosized UCA with high stability has great potential for long-term ultrasound imaging in vivo. Tumor cellular uptake and histological analysis revealed that PFP-CS-PAA nanobubbles could be taken up into tumor cells, but no intracellular uptake was observed in the case of Sonovue®. Animal fluorescence imaging in vivo demonstrated that PFP-CS-PAA nanobubbles could be effectively cleared after intravenous injection within 168 h. MTT assays indicated that PFP-CS-PAA nanobubbles had appropriate biocompatibility. Abnormal levels of blood urea nitrogen were detected after the intravenous administration of PFP-CS-PAA nanobubbles to rats, and body-weight gain was inhibited for up to 6 d, but, after that, body weights recovered their tendency to increase.