Synthesis of a novel long-circulating dual-receptor targeting and dual-modal molecular probe: an in-vitro study

Objective: To explore a novel long-circulating dual-receptor targeting and dual-modal molecular probe and investigate its physicochemical properties and targeting effect on breast cancer cells in vitro. Methods: Dual-receptor targeting and dual-modal molecular probe RGD@BBN-lipo(QDs)-SPIO was synthesized in the following steps: long-circulating liposome was prepared by film dispersion method; water-soluble superparamagnetic iron oxide (SPIO) nanoparticles and Quantum dots (QDs) were loaded in the hydrophilic and hydrophobic layer of liposome, respectively; RGD and BBN polypeptides were coupled on the former functional magnetic/fluorescent liposomes. Stability of the probe in different physiological solutions was investigated. Transmission electron microscopy (TEM) and particle size analyzer were used to measure nanoparticle sizes and the Zeta potential. Characterization of RGD and BBN was investigated through ¹H-NMR and elemental analysis. The MRI T₂ relaxivities (1/T₂) of RGD@BBN-lipo(QDs)-SPIO was measured through T₂ map scanning on 3.0 T MR system. HUV-EC-C cells were used for assessment of cells viability by MTS assay. Prussian blue staining and fluorescence imaging were carried out to determine the targeted breast cellular uptake of RGD@BBN-lipo(QDs)-SPIO nanoparticles. Results: The targeting magnetic/fluorescent dual-model molecular probes appeared spherical or para-spherical, with a mean diameter of (118.2 ± 3.9)nm, Zeta potential of (-24.78 ± 1.68) mV, MR T₂ magnetic relaxation rate of 0.4981 × 10⁶ M^-1·s^-1. RGD and BBN polypeptides were successfully coupled on the former functionally magnetic/fluorescent liposomes with the bind rates of 33.05% and 45.06%, respectively. There was low cytotoxity of the molecular probe on human umbilical vein endothelical cells (HUV-EC-C) by MTS study. Prussian blue staining and fluorescence imaging studies showed that the RGD@BBN-lipo(QDs)-SPIO nanoparticles could target any α_vβ₃ or gastrin releasing peptide receptor overexpression breast cancer. Conclusions: RGD@BBN-lipo(QDs)-SPIO is a novel long-circulating dual-receptor targeting and dual-modal molecular probe and has excellent physicochemical properties and stability, high T₂ relaxivities and strong targeting effect on cancer cells and has laid a solid foundation for early diagnosis of breast cancer. (authors)