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

Int J Nanomedicine. 2015 Sep 16:10:5805-17. doi: 10.2147/IJN.S83582. eCollection 2015.

Abstract

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; SonoVue® microbubbles; in vitro acoustic behavior; in vivo ultrasonic imaging.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Proliferation
  • Diagnostic Imaging / methods*
  • Ethylenes
  • Glioma / diagnosis*
  • Hemolysis / drug effects
  • Human Umbilical Vein Endothelial Cells / drug effects
  • Lactoferrin / chemistry*
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Micelles
  • Microbubbles*
  • Phosphates
  • Polyesters / chemistry*
  • Polymers / chemistry*
  • Rats, Sprague-Dawley
  • Tumor Cells, Cultured
  • Ultrasonics / methods*

Substances

  • Ethylenes
  • LTF protein, human
  • Micelles
  • Phosphates
  • Polyesters
  • Polymers
  • poly(lactide)
  • ethylene
  • Lactoferrin