Intrinsically Fluorescent Carbon Nanospheres as a Nuclear Targeting Vector: Delivery of Membrane-Impermeable Molecule to Modulate Gene Expression In Vivo B. Ruthrotha Selvi, †,| Dinesh Jagadeesan, ‡,| B. S. Suma, § G. Nagashankar, † M. Arif, † K. Balasubramanyam, † M. Eswaramoorthy,* ,‡ and Tapas K. Kundu* ,† Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Nanomaterials and Catalysis Laboratory, Chemistry and Physics of Materials Unit, DST Unit on Nanoscience, Confocal Facility, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for AdVanced Scientific Research, Jakkur, Bangalore-560 064, India Received May 26, 2008; Revised Manuscript Received August 19, 2008 ABSTRACT In this report, we demonstrate glucose-derived carbon nanospheres 1 to be an emerging class of intracellular carriers. The surfaces of these spheres are highly functionalized and do not need any further modification. Besides, the intrinsic fluorescence property of carbon nanospheres helps in tracking their cellular localization without any additional fluorescent tags. The spheres are found to target the nucleus of the mammalian cells, causing no toxicity. Interestingly, the in vivo experiments show that these nanospheres have an important ability to cross the blood-brain barrier and localize in the brain besides getting localized in the liver and the spleen. There is also evidence to show that they are continuously being removed from these tissues over time. Furthermore, these nanospheres were used as a carrier for the membrane-impermeable molecule CTPB (N-(4-chloro-3-trifluoromethylphenyl)-2-ethoxybenzamide), the only known small-molecule activator of histone acetyltransferase (HAT) p300. 2 Biochemical analyses such as Western blotting, immunohistochemistry, and gene expression analysis show the induction of the hyperacetylation of histone acetyltransferase (HAT) p300 (autoacetylation) as well as histones both in vitro and in vivo and the activation of HAT-dependent transcription upon CTPB delivery. These results establish an alternative path for the activation of gene expression mediated by the induction of HAT activity instead of histone deacetylase (HDAC) inhibition. The use of nanomaterials in biology has opened the door to a better understanding of cellular processes through imaging and improved therapeutic procedures, especially in drug delivery. 3 Nanoparticles such as silica, 4 LDH clay, 5 micelles, 6 polymer nanoparticles, 7 and carbon nanotubes 8,9 are being actively explored for the purpose of intracellular drug delivery. Most often, these nanoparticles require several surface chemical modifications to attach the drug molecules and additional fluorescent tags 10,11 that inevitably affect the cellular uptake and metabolism of the nanomaterial. Despite their use in transporting nucleic acids, proteins, and drug molecules across the cell membrane, the inefficiency to breach the nuclear membrane limits their use in many applications. However, nuclear targeting carriers such as peptides and polyethyleneimines face intense serum inhibi- tion as a result of their highly positive charged surfaces. 12 Here we report that carbon nanospheres (CSPs) 1 derived from the hydrothermal treatment of glucose overcome all of the above shortcomings and entered the cell nuclei. Remarkably, these CSPs are intrinsically fluorescent and do not require any additional fluorescent tags to track them inside the cells. Furthermore, the CSPs are readily dispersible in water without any prior surface modifications and could success- fully deliver a potentially therapeutic cell-impermeable molecule, CTPB, 2 inside the nucleus. Figure 1a shows the FESEM image of the 400-nm- diameter glucose-derived CSP (Supporting Information S2). The CSPs were easily dispersible in solvents such as water, ethanol, and toluene suggesting the presence of hydrophilic and hydrophobic functional groups on the surface (Figure 1b). A confocal laser scanning microscopy (CLSM) image of the CSPs was obtained by exciting the sample at 514 nm * Corresponding authors. (M.E.) E-mail: eswar@jncasr.ac.in. (T.K.K.) E-mail: tapas@jncasr.ac.in. † Transcription and Disease Laboratory, Molecular Biology and Genetics Unit. ‡ Chemistry and Physics of Materials Unit, DST Unit on Nanoscience. § Confocal Facility, Molecular Biology and Genetics Unit. | These authors contributed equally to this work. NANO LETTERS 2008 Vol. 8, No. 10 3182-3188 10.1021/nl801503m CCC: $40.75  2008 American Chemical Society Published on Web 09/19/2008