Novel lactoferrin-conjugated amphiphilic poly(aminoethyl ethylene phosphate)/poly(L-lactide) copolymer nanobubbles for tumor-targeting ultrasonic imaging

Abstract

Binhua Luo,1,2,* Huageng Liang,3,* Shengwei Zhang,3 Xiaojuan Qin,4 Xuhan Liu,1 Wei Liu,1,5 Fuqing Zeng,3 Yun Wu,6 Xiangliang Yang1,51College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 2College of Pharmacy, Hubei University of Science and Technology, Xianning, 3Department of Urology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 4Department of Medical Ultrasound, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 5National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 6Department of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo, NY, USA*These authors contributed equally to this workAbstract: In the study reported here, a novel amphiphilic poly(aminoethyl ethylene phosphate)/poly(L-lactide) (PAEEP-PLLA) copolymer was synthesized by ring-opening polymerization reaction. The perfluoropentane-filled PAEEP-PLLA nanobubbles (NBs) were prepared using the O1/O2/W double-emulsion and solvent-evaporation method, with the copolymer as the shell and liquid perfluoropentane as the core of NBs. The prepared NBs were further conjugated with lactoferrin (Lf) for tumor-cell targeting. The resulting Lf-conjugated amphiphilic poly(aminoethyl ethylene phosphate)/poly(L-lactide) nanobubbles (Lf-PAEEP-PLLA NBs) were characterized by photon correlation spectroscopy, polyacrylamide gel electrophoresis, Fourier transform infrared spectroscopy, and transmission electron microscopy. The average size of the Lf-PAEEP-PLLA NBs was 328.4±5.1 nm, with polydispersity index of 0.167±0.020, and zeta potential of -12.6±0.3 mV. Transmission electron microscopy imaging showed that the Lf-PAEEP-PLLA NBs had a near-spherical structure, were quite monodisperse, and there was a clear interface between the copolymer shell and the liquid core inside the NBs. The Lf-PAEEP-PLLA NBs also exhibited good biocompatibility in cytotoxicity and hemolysis studies and good stability during storage. The high cellular uptake of Lf-PAEEP-PLLA NBs in C6 cells (low-density lipoprotein receptor-related protein 1-positive cells) at concentrations of 0–20 µg/mL indicated that the Lf provided effective targeting for brain-tumor cells. The in vitro acoustic behavior of Lf-PAEEP-PLLA NBs was evaluated using a B-mode clinical ultrasound imaging system. In vivo ultrasound imaging was performed on tumor-bearing BALB/c nude mice, and compared with SonoVue® microbubbles, a commercial ultrasonic contrast agent. Both in vitro and in vivo ultrasound imaging indicated that the Lf-PAEEP-PLLA NBs possessed strong, long-lasting, and tumor-enhanced ultrasonic contrast ability. Taken together, these results indicate that Lf-PAEEP-PLLA NBs represent a promising nano-sized ultrasonic contrast agent for tumor-targeting ultrasonic imaging.Keywords: PAEEP-PLLA copolymer, in vitro acoustic behavior, in vivo ultrasonic imaging, SonoVue® microbubbles