Oligosaccharide‐Mediated Nuclear Transport of Nanoparticles

The transport of nanoparticles into the cellular nucleus is a potentially important technique because it can open the way to a wide range of applications, including the sequence-specific detection of genomic DNA, efficient DNA transfection, and the specific entry of drugs into the nucleus. It has been reported that the nuclear import of proteins larger than 40 kD does not occur by passive diffusion. Similarly, the nuclear import of macromolecules or particles is strictly regulated. Therefore, the nuclear import of nanoparticles that contain gold nanoparticles and quantum dots has been achieved by coating the surface with classical nuclear localization signals (NLS), that is, short, highly positively charged peptides. However, the problem remains that positively charged particles can interact with serum protein, resulting in rapid clearance from the plasma compartment. Because the cationic NLS interacts with negatively charged DNA, NLS peptides do not work as efficient signals for transport of DNA into the nucleus; this implies that the use of peptide-based cationic NLS might be limited when using DNA-displaying nanoparticles. Monsigny et al. have shown that sugars can also work as nuclear localization signals. The neoglycoproteins, BSA (bovine serum albumin)–glucose, BSA–mannose and BSA–fucose are rapidly transported into the nucleus of HeLa cells, whereas BSA without chemical modifications is not. Because carbohydrates normally show high biocompatibility and water solubility, they are suitable for use in the modification of synthetic carriers and nanoparticles. Previously, however, only the application of these carbohydrate signals to the nuclear import of proteins was examined, and there are no reports on the effectiveness of carbohydrate signals for the nuclear import of artificial materials, such as nanoparticles and polymers. Previous reports that used BSA have focused only on monosaccharides as a signal. In this paper, we expand the varieties of carbohydrates tested from monosaccharides to oligosaccharides in the search for an efficient signal that is applicable to the nuclear import of nanoparticles. Herein, we present our unique finding that nanoparticles (quantum dots) that display oligo a-glucopyranoside on their surface are readily transported into the nucleus of digitonin-permeabilized HeLa cells. Semiconductor QDs have a diameter of several nanometers and their specific transport inside the cell can be readily achieved through the display of multiple ligands on their surface. As far as we know, this is the first report to describe the import of nanoparticles into the nucleus without the use of cationic NLS. Because BSA that has been substituted with a-glucopyranoside has been reported to be efficiently transported into the cell nucleus, we synthesized neoglycolipids that contain various carbohydrates comprised of different numbers of glucose units (Scheme 1). In addition to a-monoglucopyranoside–lipid 3, we synthesized maltose(Glca1-4Glc)–lipid 4, maltotriose(Glca1-4Glca1-4Glc)–lipid 5, and panose(Glca1-6Glca1-4Glc)–lipid 6. The cellotriose(Glcb1-4Glcb1-4Glc)–lipid 7 is a trisaccharide that is composed of only a b-linked glucose, thus lipid 7 was used as a control to a-linked glucose. Neoglycolipids were synthesized based on a previously described method 11] by starting from fully acetylated carbohydrates, hexa(ethyleneglycol) and 11-bromoundecene. CdTe nanocrystals that had been stabilized with mercaptopropionic acid (MPA) were prepared in water as described by Yang et al. Sugar-displaying CdTe QDs were synthesized by surface exchange from MPA to the neoglycolipid in water (Figure 1A), and then purified by using spin filtration. Ligand exchange occurring on the surface of the QDs was confirmed by MALDI-ToF mass spectrometry. Ligands immobilized on inorganic nanoparticles, such as QDs, were detached from the surface during the laser deposition process, and the mass corresponding to the molecular weight of neoglycolipids was clearly detected (Figure 1B). Furthermore, the display of sugars on the QDs was visually confirmed by trapping on a lectin-immobilized column. For example, maltotriose 5–QDs were specifically trapped on a ConA (concanavalin A; a-mannose and a-glucose specific) agarose column, whereas they were not trapped on WGA (wheat germ agglutinin; GlcNAc specific) or LCA (lens culinaris agglutinin; branched-fucose specific) agarose columns (data not shown). We explored the interactions between sugar-displaying CdTe QDs and digitonin-permeabilized HeLa cells. Digitonin treatment causes partial damage to the plasma membrane and increases the permeability of cells. Digitonin permeabilization has been used often in the study of biochemical processes that are related to the import and export of nuclear proteins. Although live cells become semi-intact upon digitonin treatment due to the leakage of cytoplasmic proteins through the plasma membrane, the nuclear membrane is left intact. Import buffer (pH 7.3, 20 mm HEPES, 110 mm KOAc, 5 mm NaOAc, 2 mm MgACHTUNGTRENNUNG(OAc)2, 0.5 mm EGTA) that contained the sugar-displaying CdTe QDs (0.6 mgmL , [a] Dr. K. Niikura, Dr. Y. Matsuo, Dr. K. Ijiro Research Institute for Electronic Science, Hokkaido University N21W10, Kita-ku, Sapporo 001-0021 (Japan) Fax: (+81)11-706-9361 E-mail : kniikura@poly.es.hokudai.ac.jp ijiro@poly.es.hokudai.ac.jp [b] S. Sekiguchi, T. Nishio Department of Chemistry, Hokkaido University N21W10, Kita-ku, Sapporo 001-0021 (Japan) [c] T. Masuda, Dr. H. Akita, Dr. H. Harashima Faculty of Pharmaceutical Science, Hokkaido University N12 W6, Kita-Ku, Sapporo 060-0812 (Japan) [d] Dr. K. Kogure Department of Biophysical Chemistry, Kyoto Pharmaceutical University Misasagi-Nakauchicho 5, Yamashinaku, Kyoto 607-84142 (Japan)