Pentapartite fractionation of particles in oral fluids by differential centrifugation.

Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers. For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Canonical exosome markers, including CD63, CD9, CD133, and HSP70, were found in all fractions, whereas CD81 and AQP5 were enriched in the 160K fraction, with non-negligible amounts in the 2K fraction. The 2K fraction also contained its characteristic markers that included short derivatives of EGFR and E-cadherin, as well as an autophagosome marker, LC3, and large multi-layered vesicles were observed by electronic microscopy. Most of the DNA and RNA was recovered from the 0.3K and 2K fractions, with some in the 160K fraction. These results can provide guideline information for development of purpose-designed OF-based diagnostic systems.

Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine.

Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes - or, more properly, small extracellular vesicles (sEVs) - which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (-28 mV vs. -21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy.

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.

Immobilization of a carbon nanomaterial-based localized drug-release system using a bispecific material-binding peptide.

INTRODUCTION: Inorganic materials are widely used in medical devices, such as artificial hearts, vessels, and joints, in stents, and as nanocarriers for drug-delivery systems. Carbon nanomaterials are of particular interest due to their biological inertness and their capability to accommodate molecules. Several attempts have been proposed, in which carbon nanomaterials are used as nanocarriers for the systemic delivery of drugs. MATERIALS AND METHODS: We developed a drug-delivery system in which oxidized single-walled carbon nanohorns (oxSWNHs) were immobilized on a titanium (Ti) surface using material-binding peptides to enable localized drug delivery. For this purpose, we utilized a bispecific peptidic aptamer comprising a core sequence of a Ti-binding peptide and a SWNH-binding peptide to immobilize oxSWNHs on Ti. RESULTS: Scanning electron microscopy was used to confirm the presence of oxSWNHs adsorbed onto the Ti surface, and a quartz crystal microbalance was used to evaluate the binding process during oxSWNH adsorption. The oxSWNHs-ornamented Ti substrate was nontoxic to cells and released biologically active dexamethasone over a sustained period. CONCLUSION: This oxSWNHs-immobilized system can be used to modify the surface of Ti in implants and be loaded with drugs that stimulate osteogenesis and bone regeneration.

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.

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.

Not nanocarbon but dispersant induced abnormality in lysosome in macrophages in vivo.

The properties of nanocarbons change from hydrophobic to hydrophilic as a result of coating them with dispersants, typically phospholipid polyethylene glycols, for biological studies. It has been shown that the dispersants remain attached to the nanocarbons when they are injected in mice and influence the nanocarbons' biodistribution in vivo. We show in this report that the effects of dispersants also appear at the subcellular level in vivo. Carbon nanohorns (CNHs), a type of nanocarbon, were dispersed with ceramide polyethylene glycol (CPEG) and intravenously injected in mice. Histological observations and electron microscopy with energy dispersive x-ray analysis revealed that, in liver and spleen, the lysosome membranes were damaged, and the nanohorns formed a complex with hemosiderin in the lysosomes of the macrophages. It is inferred that the lysosomal membrane was damaged by sphigosine generated as a result of CPEG decomposition, which changed the intra lysosomal conditions, inducing the formation of the CPEG-CNH and hemosiderin complex. For comparison, when glucose was used instead of CPEG, neither the nanohorn­hemosiderin complex nor lysosomal membrane damage was found. Our results suggest that surface functionalization can control the behavior of nancarbons in cells in vivo and thereby improve their suitability for medical applications.

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.

A tumor-environment-responsive nanocarrier that evolves its surface properties upon sensing matrix metalloproteinase-2 and initiates agglomeration to enhance T2 relaxivity for magnetic resonance imaging.

We designed and synthesized a modified ferritin as a tumor-environment-responsive nanocarrier. We found that this nanocarrier could evolve its surface properties upon sensing a tumor-associated protease, matrix metalloproteinase-2 (MMP-2), which initiated agglomeration, resulting in the enhancement of T(2) relaxivity for magnetic resonance imaging (MRI). The designed ferritin contained a triad of modifiers composed of (i) a "sensing" segment (substrate peptide of MMP-2), (ii) "hydrophobic" segments and (iii) a "hydrophilic" segment of polyethylene glycol (PEG). The hydrophilic segment ensured the particles' monodispersibility in aqueous conditions. In the presence of MMP-2 activity, the "sensing" segment was cleaved by the enzyme and its submerged "hydrophobic" segments were exposed on the surface, resulting in the initiation of aggregation. Because ferritin contains ferrihydrite in its inner space, this multimerization resulted in the enhancement of T(2) relaxivity, suggesting that this nanocarrier may be useful as a contrast agent in MRI.

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.

Peptide nanofibers modified with a protein by using designed anchor molecules bearing hydrophobic and functional moieties.

Self-assembly of peptides and proteins is a key feature of biological functions. Short amphiphilic peptides designed with a beta-sheet structure can form sophisticated nanofiber structures, and the fibers are available as nanomaterials for arranging biomolecules. Peptide FI (H-PKFKIIEFEP-OH) self-assembles into nanofibers with a coiled fine structure, as reported in our previous work. We have constructed anchor molecules that have both a binding moiety for the fiber structure and a functional unit capable of capturing target molecules, with the purpose of arranging proteins on the designed peptide nanofibers. Designed anchors containing an alkyl chain as a binding unit and biotin as a functional moiety were found to bind to peptide fibers FI and F2i (H-ALEAKFAAFEAKLA-NH(2)). The surface-exposed biotin moiety on the fibers could capture an anti-biotin antibody. Moreover, hydrophobic dipeptide anchor units composed of iminodiacetate connected to Phe-Phe or Ile-Ile and a peptide composed of six histidine residues connected to biotin could also connect FI peptide fibers to the anti-biotin antibody through the chelation of Ni(2+) ions. This strategy of using designed anchors opens a novel approach to constructing nanoscale protein arrays on peptide nanomaterials.

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.

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.

Dispersion of cisplatin-loaded carbon nanohorns with a conjugate comprised of an artificial peptide aptamer and polyethylene glycol.

Hydrophobic single-wall carbon nanohorns (SWNHs) were dispersed in aqueous media by noncovalently modifying their surfaces with conjugate molecules comprised of polyethylene glycol (PEG) and a peptide aptamer (NHBP-1) that specifically bind to the surfaces of the SWNHs. The conjugates were synthesized by coupling PEG (average molecular weights of 20,000 and 5000) to the N-terminus of NHBP-1 to produce 20PEG-NHBP and 5PEG-NHBP, respectively. Oxidized SWNHs (oxSWNHs) mixed with 20PEG-NHBP or 5PEG-NHBP were well dispersed in water and passed through a gel filtration column, whereas the oxSWNHs treated with PEG stuck to the top of the column. Although the presence of salts in the media significantly impaired the dispersibility of the oxSWNHs, the oxSWNHs/20PEG-NHBP complexes were well dispersed in both the phosphate-buffered saline (PBS) and cell culture medium. The amount of 20PEG-NHBP bound to the oxSWNHs was estimated to be 0.32 g/g of oxSWNHs, and a dynamic light scattering analysis revealed the diameter of the oxSWNHs/20PEG-NHBP complex to be approximately 210 nm. We then showed that CDDP@oxSWNHs/20PEG-NHBP, in which the cancer chemotherapy drug cisplatin (CDDP) was loaded inside the oxSWNHs, was well dispersed in both the PBS and culture medium and exerted a potent cytotoxic effect against cancer cells. The good dispersion of drug-loaded carbon nanomaterials, like that seen here, is a prerequisite for the clinical application of such materials.

Design of peptides that form amyloid-like fibrils capturing amyloid beta1-42 peptides.

Amyloid beta-peptide (Abeta) plays a critical role in Alzheimer's disease (AD). The monomeric state of Abeta can self-assemble into oligomers, protofibrils, and amyloid fibrils. Since the fibrils and soluble oligomers are believed to be responsible for AD, the construction of molecules capable of capturing these species could prove valuable as a means of detecting these potentially toxic species and of providing information pertinent for designing drugs effective against AD. To this aim, we have designed short peptides with various hydrophobicities based on the sequence of Abeta14-23, which is a critical region for amyloid fibril formation. The binding of the designed peptides to Abeta and the amplification of the formation of peptide amyloid-like fibrils coassembled with Abeta are elucidated. A fluorescence assay utilizing thioflavin T, known to bind specifically to amyloid fibrils, revealed that two designed peptides (LF and VF, with the leucine and valine residues, respectively, in the hydrophobic core region) could form amyloid-like fibrils effectively by using mature Abeta1-42 fibrils as nuclei. Peptide LF also coassembled with soluble Abeta oligomers into peptide fibrils. Various analyses, including immunostaining with gold nanoparticles, enzyme-linked immunosorbent assays, and size-exclusion chromatography, confirmed that the LF and VF peptides formed amyloid-like fibrils by capturing and incorporating Abeta1-42 aggregates into their peptide fibrils. In this system, small amounts of mature Abeta1-42 fibrils or soluble oligomers could be transformed into peptide fibrils and detected by amplifying the amyloid-like fibrils with the designed peptides.

Construction of biotinylated peptide nanotubes for arranging proteins.

Three kinds of biotinylated peptides with different linkers between biotin and beta-sheet peptide were designed and synthesized. The transmission electron microscopy revealed that the biotinylated peptides self-assembled to form a tubular structure with external diameter of ca. 60 nm and inner diameter of ca. 30 nm in an aqueous solution. The anti-biotin antibody effectively bound to biotin groups in the peptide nanotubes. The binding of antibody was regulated by not only the concentration of the protein in the solution but also the properties of biotinylated peptides forming the tubes. The antibody preferentially bound to the biotinylated peptide tubes assembled from the peptide with the most hydrophilic linker, suggesting that the surface properties and functions of the tubular structure were modulated and engineered by the design of the peptides.

Construction of a protein array on amyloid-like fibrils using co-assembly of designed peptides.

Amyloid-like fibrils formed from de novo designed short peptides, made up a nanoscale scaffold on which streptavidin was arranged in a regular spacing, potentially allowing the development into an array technology utilizing bio-nanoconstructs.

Fabrication of nanofibers with uniform morphology by self-assembly of designed peptides.

Fabrication of controlled peptide nanofibers with homogeneous morphology has been demonstrated. Amphiphilic beta-sheet peptides were designed as sequences of Pro-Lys-X(1)-Lys-X(2)-X(2)-Glu-X(1)-Glu-Pro. X(1) and X(2) were hydrophobic residues selected from Phe, Ile, Val, or Tyr. The peptide FI (X(1)=Phe; X(2)=Ile) self-assemble into straight fibers with 80-120 nm widths and clear edges, as examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The fiber formation is performed in a hierarchical manner: beta-sheet peptides form a protofibril, the protofibrils assemble side-by-side to form a ribbon, and the ribbons then coil in a left-handed fashion to make up a straight fiber. These type of fibers are formed from peptides possessing hydrophobic aromatic Phe residue(s). Furthermore, a peptide with Ala residues at both N and C termini does not form fibers (100 nm scale) with clear edges; this causes random aggregation of small pieces of fibers instead. Thus, the combination of unique amphiphilic sequences and terminal Pro residues determine the fiber morphology.

Complementary nucleobase interaction enhances peptide-peptide recognition and self-replicating catalysis.

The availability of the complementary interaction of nucleobases for influencing the formation of peptide architectures was explored. Nucleobases were incorporated as additional recognition elements in coiled-coil peptides by employing nucleobase amino acids (NBAs), which are artificial L-alpha-amino gamma-nucleobase-butyric acids. The effect of the base-pair interaction on intermolecular recognition between peptides was evaluated through a self-replication reaction. The self-replication reactions of the peptides with complementary base pairs such as thymine-adenine or guanine-cytosine at the g-g' heptad positions were accelerated in comparison with those of the peptides with mismatched base pairs or without nucleobases. However, thymine-adenine pairs at the e-e' positions did not enhance the self-replication. In the presence of a denaturant, the enhancement effects of complementary base pairs on the reaction disappeared. Thermal denaturation studies showed that the thymine-adenine pairs contributed to stabilization of the coiled-coil structure and that the pairs at the g-g' positions were more effective than those at the e-e' positions. The peptide-peptide interaction was reinforced by complementary nucleobase interactions appropriately arranged in the peptide structure; these led to acceleration of the self-replication reactions.