[Preparation of folate-targeted magnetic nanocomposites loaded with TFPI-2 plasmid and cisplatin and evaluation of its targeting and inhibitory effect on nasopharyngeal carcinoma HNE-1 cells in vitro].

OBJECTIVE:To prepare a novel folate-targeted magnetic nanocomposites loaded with tissue facor pathway inhibitor 2 (TFPI-2) and cisplatin (CDDP) and to investigate its targeting ability and anti-tumor effect on nasopharyngeal carcinoma HNE-1 cells in vitro. METHODS:The copolymer folic acid-polyethylene glycol-polyethyleneimine (FA-PEG-PEI) was synthesized through amidation reaction, and then FA-PEG-PEI/ magnetic nanoparticles-CDDP/TFPI-2 (MNP-CDDP/TFPI-2) nanocomposites was obtained by electrostatic adsorption between TFPI-2 plasmid and magnetic nanoparticles loaded with CDDP (MNP-CDDP) with vortex FA-PEG-PEI. (1)H Nuclear Magnetic Resonance ((1)H NMR ) was used to determine if FA-PEG-PEI was synthesized. The particle size, zeta potential and morphology were detected by dynamic light scattering (DLS) and transmission electron microscope (TEM). The content of Fe and CDDP was measured by phenanthroline and o-phenylenediamine (OPDA) colourimetry. Agarose gel electrophoresis was used to analyze the binding ability of FA-PEG-PEI/MNP-CDDP to TFPI-2 plasmid. Molecular targeted uptake of FA-PEG-PEI/ MNP-CDDP/TFPI-2 coupling with green fluorescent protein (GFP) in NPC cells were observed by Prussian-blue iron staining and fluorescence microscope. The levels of TFPI-2 protein expression after transfection were evaluated by Western blot. The effects of nanocomposites on HNE-1 cells proliferation and apoptosis were measured with Cell Counting Kit-8(CCK-8) and flow cytometry. RESULTS:Special peak value of FA, PEG and PEI were showed on (1)H NMR spectrogram. The mean size and zeta potential of FA-PEG-PEI/MNP-CDDP/TFPI-2 were 141.1 nm and 21.5 mV. The nanocomposites showed a good monodispersity and an insufficient size uniformity under TEM. The content of Fe and CDDP were 116.2 μg/ml and 92.88 μg/ml, respectively. Agarose gel electrophoresis showed TFPI-2 could be encapsulated completely and protected from digestion of DNA enzyme as the mass ratio of FA-PEG-PEI/ MNP-CDDP and TFPI-2 plasmid was equal or higher than 1∶1. More blue-stained magnetic granulars and green fluorescence were seen in folate receptor (FR)-positive HNE-1 cells than in FR-negative CNE-2 (P<0.05) under microscope and fluorescence microscope. The level of TFPI-2 protein expression in HNE-1cells increased significantly after transfection by FA-PEG-PEI/ MNP-CDDP/TFPI-2, compared with other control groups (FA-PEG-PEI/MNP-CDDP group and TFPI-2 group), all P<0.05. The nanocomposites inhibitory effect on HNE-1 including cell growth inhibition rate (64.00%) and apoptosis rate (49.61%) were significantly higher than that in FA-PEG-PEI/MNP group (8.19%, 9.26%), FA-PEG-PEI/TFPI-2 group (40.35%, 19.85%) and FA-PEG-PEI/MNP-CDDP group(56.15%, 36.46%)(P<0.05). CONCLUSION:FA-PEG-PEI/MNP-CDDP/TFPI-2 nanocomposites was successfully synthesized using amidation and electrostatic adsorption technology and has a good molecular targeting and inhibitory effect on FR-positive HNE-1cells in vitro.