Fundamental understanding of how crystals of organic molecules nucleate on a surface remains limited because of the difficulty of probing rare events at the molecular scale. Here we show that single-molecule templates on the surface of carbon nanohorns can nucleate the crystallization of two organic compounds from a supersaturated solution by mediating the formation of disordered and mobile molecular nanoclusters on the templates. Single-molecule real-time transmission electron microscopy indicates that each nanocluster consists of a maximum of approximately 15 molecules, that there are fewer nanoclusters than crystals in solution, and that in the absence of templates physisorption, but not crystal formation, occurs. Our findings suggest that template-induced heterogeneous nucleation mechanistically resembles two-step homogeneous nucleation.

Application of nanotechnology to medical biology has brought remarkable success. Water-soluble fullerenes are molecules with great potential for biological use because they can endow unique characteristics of amphipathic property and form a self-assembled structure by chemical modification. Effective gene delivery in vitro with tetra(piperazino)fullerene epoxide (TPFE) and its superiority to Lipofectin have been described in a previous report. For this study, we evaluated the efficacy of in vivo gene delivery by TPFE. Delivery of enhanced green fluorescent protein gene (EGFP) by TPFE on pregnant female ICR mice showed distinct organ selectivity compared with Lipofectin; moreover, higher gene expression by TPFE was found in liver and spleen, but not in the lung. No acute toxicity of TPFE was found for the liver and kidney, although Lipofectin significantly increased liver enzymes and blood urea nitrogen. In fetal tissues, neither TPFE nor Lipofectin induced EGFP gene expression. Delivery of insulin 2 gene to female C57/BL6 mice increased plasma insulin levels and reduced blood glucose concentrations, indicating the potential of TPFE-based gene delivery for clinical application. In conclusion, this study demonstrated effective gene delivery in vivo for the first time using a water-soluble fullerene.

Twenty potassium complexes of penta-[(4-substituted)phenyl][60]fullerene anions were synthesized and examined for their ability to form bilayer vesicles in water. The 4-substituents include alkyl groups ranging from methyl to icosanyl groups and perfluoromethyl, perfluorobutyl, and perfluorooctyl groups. The overall structure of the amphiphiles can be described as a nonpolar/polar/nonpolar (n-p-n') motif as opposed to the usual polar/nonpolar motif of lipid amphiphiles. Despite the hydrophobicity of the fullerene moiety (n-part) and alkyl/perfluoroalkyl chains (n'-part), all compounds except for the one with perfluoromethyl groups were soluble in water because of the centrally located fullerene cyclopentadienide (p-part) and spontaneously formed a vesicle of 25- to 60-nm diameter with a narrow unimodal size distribution. The vesicles are stable upon heating to 90 °C or standing over one year in air, as well as on a solid substrate in air or in vacuum, maintaining their spherical form. The vesicle membrane consists of an interdigitated bilayer of the amphiphile molecules, in which the fullerene n-part is inside and the n'-side is exposed to water. These vesicles, in particular the one bearing icosanyl chains, exhibit the smallest water permeability coefficient ever found for a self-assembled membrane in water.

Lipid bilayer membranes are important as fundamental structures in biology and possess characteristic water-permeability, stability, and mechanical properties. Water permeation through a lipid bilayer membrane occurs readily, and more readily at higher temperature, which is largely due to an enthalpy cost of the liquid-to-gas phase transition of water. A fullerene bilayer membrane formed by dissolution of a water-soluble fullerene, Ph 5C60K, has now been shown to possess properties entirely different from those of the lipid membranes. The fullerene membrane is several orders of magnitude less permeable to water than a lipid membrane, and the permeability decreases at higher temperature. Water permeation is burdened by a very large entropy loss and may be favored slightly by an enthalpy gain, which is contrary to the energetics observed for the lipid membrane. We ascribe this energetics to favorable interactions of water molecules to the surface of the fullerene molecules as they pass through the clefts of the rigid fullerene bilayer. The findings provide possibilities of membrane design in science and technology.bilayer membrane ͉ vesicle ͉ entropy barrier V esicles made of a lipid bilayer membrane such as a cell membrane are fundamental structures in biology. The membrane maintains stability and mechanical properties to sustain the integrity of the biological machinery while allowing water to permeate through the liquid crystalline film of the hydrocarbon chains (1). The low energy barrier for water permeation is largely due to the small enthalpy cost of the liquid-to-gas phase transition of water (2). The lipid membrane and its water permeation mechanism have long served as important models for design and analysis of membrane structures. We have reported that, upon dissolution in water, water-soluble fullerene (Ph 5 C 60 K) ( Fig. 1) (3) produces spherical bilayer vesicles (Ϸ15-nm radius) [see supporting information (SI) Fig. 7] as an exclusive self-assembly object (4, 5). We report here that the vesicles made from water-soluble fullerene molecules show water-permeability, stability, and mechanical characteristics entirely different from those of the conventional vesicles. The fullerene membrane is several orders of magnitude less permeable to water than a lipid membrane (2, 6, 7), and the permeability decreases at higher temperature. The high energy barrier for permeation is due to a very large negative activation entropy. We suggest that water molecules lose their mobility in the clefts of the rigid fullerene bilayer, just as in hydration of crystalline proteins or metal salts. Results and DiscussionA solution study of the thermodynamics of water permeation through the fullerene membrane revealed that the mechanism of permeation is very different from that so far known for lipid bilayers. The vesicles were found to be quite tough on a mica surface, as examined by atomic force microscopy (4). In contrast to lipid vesicles, the fullerene vesicles show neither 1 H nor 13 C NMR signals in water (at 10 -80°C), an...