Evaluation of Complex Formation between Aminated Polyrotaxane and Proteins Using FRET

We previously developed an intracellular carrier, an aminated polyrotaxane (amino-PRX), that recognizes and deforms in response to the shape and charge distribution of proteins and nucleic acids. Amino-PRX can deliver various biopharmaceuticals into cells with high efficiency. However, the detailed intracellular behavior of the carrier alone and its complexes in the cells remains unclear. In addition, the usefulness of amino-PRX as a carrier for introducing antibodies into cells is unclear. In this study, we prepared fluorescently capped amino-PRX to visualize the intracellular distribution of the carrier, drugs, and the complexes. Moreover, not only the fluorescence of the carrier and drug themselves within cells but also Förster resonance energy transfer (FRET)-derived fluorescence between the two components was observed to examine the intracellular behavior of the complex. First, fluorescently capped PRX was prepared by capping polypseudorotaxane, consisting of α-cyclodextrin (α-CyD) and polyethylene glycol (2 or 20 kDa), with 5(6)-carboxyfluorescein (FAM). Next, diethylenetriamine (DET) was modified onto the α-CyD of PRX to obtain amino-PRX, namely, DET-PRX_-FAM (2 or 20 kDa). In HeLa cells, DET-PRX_-FAM (2 kDa) exhibited lower cytotoxicity compared to DET-PRX_-FAM (20 kDa). Importantly, FRET-derived fluorescence was observed upon adding DET-PRX_-FAM (2 kDa) to phycocyanin or HiLyte Fluor™ 647-modified human immunoglobulin G due to complex formation. Moreover, fluorescence from the carrier, proteins, and FRET, derived from the complex, was observed in HeLa cells. These results suggest the successful construction of a fluorescent molecular-capped amino-PRX that enables visualization of the intracellular distribution of the carrier, drugs, and the complexes.