Sachiko Matsumura

Biodistribution and Ultrastructural Localization of Single-Walled Carbon Nanohorns Determined In Vivo with Embedded Gd2O3 Labels

Single-walled carbon nanohorns (SWNHs) are single-graphene tubules that have shown high potential for drug delivery systems. In drug delivery, it is essential to quantitatively determine biodistribution and ultrastructural localization. However, to date, these determinations have not been successfully achieved. In this report, we describe for the first time a method that can achieve these determinations. We embedded Gd(2)O(3) nanoparticles within SWNH aggregates (Gd(2)O(3)@SWNHag) to facilitate detection and quantification. Gd(2)O(3)@SWNHag was intravenously injected into mice, and the quantities of Gd in the internal organs were measured by inductively coupled plasma atomic emission spectroscopy: 70-80% of the total injected material accumulated in liver. The high electron scattering ability of Gd allows detection with energy dispersive X-ray spectroscopy and facilitates the ultrastructural localization of individual Gd(2)O(3)@SWNHag with transmission electron microscopy. In the liver, we found that the Gd(2)O(3)@SWNHag was localized in Kupffer cells but were not observed in hepatocytes. In the Kupffer cells, most of the Gd(2)O(3)@SWNHag was detected inside phagosomes, but some were in another cytoplasmic compartment that was most likely the phagolysosome.

Peptide-coated, self-assembled M12L24 coordination spheres and their immobilization onto an inorganic surface

A nano-sized M12L24 coordination sphere coated with 24 hexapeptide aptamers (Arg–Lys–Leu–Pro–Asp–Ala: minTBP-1) was efficiently self-assembled from 12 Pd(II) ions and 24 bent ligands with minTBP-1 at their convex. The high density of aptamers covering the 3.5 nm diameter sphere resulted in irreversible immobilization on a Ti surface, in contrast to the relatively weak, reversible binding of a single aptamer.

Prevention of carbon nanohorn agglomeration using a conjugate composed of comb-shaped polyethylene glycol and a peptide aptamer.

Assured dispersibility is a prerequisite for clinical application of nanomaterials. Carbon nanomaterials have hydrophobic surfaces and thus readily agglomerate under aqueous conditions. Various conjugates composed of a carbon surface-binding moiety and polyethylene glycol (PEG) have been examined as dispersants for carbon nanomaterials. Here we synthesized a conjugate composed of a comb-shaped PEG (cPEG) and carbon nanomaterial-binding peptide (NHBP-1). The resultant cPEG-NHBP3 conjugate displayed multiple units (2.4 on average) of NHBP-1 on a single cPEG molecule whose average molecular weight was 15-20 kDa. cPEG-NHBP3 endowed single-walled carbon nanohorns (SWNHs) with good dispersibility in vitro, which could not be achieved with 20PEG-NHBP, a conjugate composed of linear 20 kDa PEG and a single NHBP-1 peptide. Notably, cPEG-NHBP1, which was similar to 20PEG-NHBP but had a comb-shaped PEG backbone, functioned better as a dispersant than 20PEG-NHBP, suggesting a graft-type PEG formula is better-suited for dispersing nanomaterials. Finally, cPEG-NHBP3 treatment substantially suppressed formation of SWNH agglomerates in mouse lung, suggesting the potential utility of SWNHs as a carrier in drug delivery systems.

Bridging Adhesion of a Protein onto an Inorganic Surface Using Self-Assembled Dual-Functionalized Spheres

For the bridging adhesion of different classes of materials in their intact functional states, the adhesion of biomolecules onto inorganic surfaces is a necessity. A new molecular design strategy for bridging adhesion was demonstrated by the introduction of two independent recognition groups on the periphery of spherical complexes self-assembled from metal ions (M) and bidentate ligands (L). These dual-functionalized M12L24 spheres were quantitatively synthesized in one step from two ligands, bearing either a biotin for streptavidin recognition or a titania-binding aptamer, and Pd(II) ions. The selective recognition of titania surfaces was achieved by ligands with hexapeptide aptamers (Arg-Lys-Leu-Pro-Asp-Ala: minTBP-1), whose fixation ability was enhanced by the accumulation effect on the surface of the M12L24 spheres. These well-defined spherical structures can be specifically tailored to promote interactions with both titania and streptavidin simultaneously without detrimentally affecting either recognition motif. The irreversible immobilization of the spheres onto titania was revealed quantitatively by quartz crystal microbalance measurements, and the adhesion of streptavidin to the titania surface mediated by the biotin surrounding the spheres was visually demonstrated by lithographic patterning experiments.

Carbon nanohorns accelerate bone regeneration in rat calvarial bone defect

A recent study showed that carbon nanohorns (CNHs) have biocompatibility and possible medical uses such as in drug delivery systems. It was reported that some kinds of carbon nanomaterials such as carbon nanotubes were useful for bone formation. However, the effect of CNHs on bone tissue has not been clarified. The purpose of this study was to evaluate the effect of CNHs on bone regeneration and their possible application for guided bone regeneration (GBR). CNHs dispersed in ethanol were fixed on a porous polytetrafluoroethylene membrane by vacuum filtration. Cranial defects were created in rats and covered by a membrane with/without CNHs. At two weeks, bone formation under the membrane with CNHs had progressed more than under that without CNHs and numerous macrophages were observed attached to CNHs. At eight weeks, there was no significant difference in the amount of newly formed bone between the groups and the appearance of macrophages was decreased compared with that at two weeks. Newly formed bone attached to some CNHs directly. These results suggest that macrophages induced by CNHs are related to bone regeneration. In conclusion, the present study indicates that CNHs are compatible with bone tissue and effective as a material for GBR.

Ultrastructural localization of intravenously injected carbon nanohorns in tumor

Nanocarbons have many potential medical applications. Drug delivery, diagnostic imaging, and photohyperthermia therapy, especially in the treatment of tumors, have attracted interest. For the further advancement of these application studies, the microscopic localization of nanocarbons in tumor tissues and cells is a prerequisite. In this study, carbon nanohorns (CNHs) with sizes of about 100 nm were intravenously injected into mice having subcutaneously transplanted tumors, and the CNHs in tumor tissue were observed with optical and electron microscopy. In the tumor tissue, the CNHs were found in macrophages and endothelial cells within the blood vessels. Few CNHs were found in tumor cells or in the region away from blood vessels, suggesting that, under these study conditions, the enhanced permeability of tumor blood vessels was not effective for the movement of CNHs through the vessel walls. The CNHs in normal skin tissue were similarly observed. The extravasation of CNHs was not so obvious in tumor but was easily found in normal skin, which was probably due to their vessel wall structure difference. Proper understanding of the location of CNHs in tissues is helpful in the development of the medical uses of CNHs.

Preferential capture of EpCAM-expressing extracellular vesicles on solid surfaces coated with an aptamer-conjugated zwitterionic polymer

Extracellular vesicles (EVs) collectively represent small vesicles that are secreted from cells and carry biomolecules (e.g., miRNA, lncRNA, mRNA, proteins, lipids, metabolites, etc.) that originate in those cells. Body fluids, such as blood and saliva, include large numbers of EVs, making them potentially a rich source of diagnostic information. However, these EVs are mixtures of vesicles released from diseased tissues as well as from normal cells. This heterogeneous nature therefore blurs the clinical information obtainable from EV‐based diagnosis. Here, we synthesized an EpCAM‐affinity coating agent, which consists of a peptide aptamer for EpCAM and a zwitterionic MPC polymer, and have shown that this conjugate endowed the surfaces of inorganic materials with the preferential affinity to EpCAM‐expressing EVs. This coating agent, designated as EpiVeta, could be useful as a coating for various diagnostic devices to allow concentration of cancer‐related EVs from heterogeneous EV mixtures.

Host Cell Prediction of Exosomes Using Morphological Features on Solid Surfaces Analyzed by Machine Learning

Exosomes are extracellular nanovesicles released from any cells and found in any body fluid. Because exosomes exhibit information of their host cells (secreting cells), their analysis is expected to be a powerful tool for early diagnosis of cancers. To predict the host cells, we extracted multidimensional feature data about size, shape, and deformation of exosomes immobilized on solid surfaces by atomic force microscopy (AFM). The key idea is combination of support vector machine (SVM) learning for individual exosome particles and their interpretation by principal component analysis (PCA). We observed exosomes derived from three different cancer cells on SiO2/Si, 3-aminopropyltriethoxysilane-modified-SiO2/Si, and TiO2 substrates by AFM. Then, 14-dimensional feature vectors were extracted from AFM particle data, and classifiers were trained in 14-dimensional space. The prediction accuracy for host cells of test AFM particles was examined by the cross-validation test. As a result, we obtained prediction of exosome host cells with the best accuracy of 85.2% for two-class SVM learning and 82.6% for three-class one. By PCA of the particle classifiers, we concluded that the main factors for prediction accuracy and its strong dependence on substrates are incremental decrease in the PCA-defined aspect ratio of the particles with their volume.