Effects of Tat peptide on intracellular delivery of arsenic trioxide albumin microspheres.

ABSTRACT

The current study was designed to evaluate the ability of cell-penetrating peptides to deliver arsenic trioxide albumin microspheres (AsAMs) into bladder cancer cells. The transactivating transcriptional activator (Tat) peptide was labeled with the enhanced green fluorescent protein (EGFP) using eukaryotic vector construction and fusion gene expression techniques. Arsenic trioxide albumin mirospheres were prepared using the chemical crosslink and solidification method. The conjugate, Tat-EGFP-As2O3-AMs (TEAsAMs), was synthesized using the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio) propionate and verified by electrophoresis under reducing conditions and fluorescence microscopy. The intracellular delivery of TEAsAMs was evaluated by laser confocal microscopy and transmission electron microscopy. The arsenic content in the bladder cancer EJ cells was assayed to evaluate the efficiency of delivery. Gene sequencing showed that the pET-Tat-EGFP expression vector was constructed successfully. The expression of the Tat-EGFP fusion protein was verified by matrix-assisted laser desorption/ionization-time of flight analysis, and the protein was transduced into cell cytoplasm as observed under a fluorescence microscope. Electrophoresis under reducing conditions demonstrated the covalent linkage between Tat-EGFP and AsAMs. Under a laser confocal microscope and a transmission electron microscope, TEAsAMs surrounded by green fluorescence were shown to enter the cells faster than EGFP-As2O3-AMs, with an increase in the intracellular arsenic content being observed in cells treated with TEAsAMs compared with those treated with EGFP-As2O3-AMs. These results suggest that Tat peptide promotes the cellular uptake of large albumin microspheres with encapsulated arsenicals.