CD63 sorts cholesterol into endosomes for storage and distribution via exosomes.

Extracellular vesicles such as exosomes are now recognized as key players in intercellular communication. Their role is influenced by the specific repertoires of proteins and lipids, which are enriched when they are generated as intraluminal vesicles (ILVs) in multivesicular endosomes. Here we report that a key component of small extracellular vesicles, the tetraspanin CD63, sorts cholesterol to ILVs, generating a pool that can be mobilized by the NPC1/2 complex, and exported via exosomes to recipient cells. In the absence of CD63, cholesterol is retrieved from the endosomes by actin-dependent vesicular transport, placing CD63 and cholesterol at the centre of a balance between inward and outward budding of endomembranes. These results establish CD63 as a lipid-sorting mechanism within endosomes, and show that ILVs and exosomes are alternative providers of cholesterol.

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties.

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.

Syndiotactic hexamer peptide nanodots.

Spatial confinement of excitons in the nano-crystalline region of semiconducting nanostructures differ significantly from the optoelectronic properties exhibited by the bulk material. We report spike-like absorption observed in the UV spectrum of a phenylalanine hexamer peptide [(Ff)3-OH] nano-assembly, which may be attributed to the spatial confinement of electrons to the dimension of quantum dots. Interdependency of the UV and PLE spectrum of the peptide confirms the existence of quantum confinement in (Ff)3-OH nano-assemblies.

Aromatic interactions directing peptide nano-assembly.

Self-assembly is a process of spontaneous organization of molecules as a result of non-covalent interactions. Organized self-assembly at the nano level is emerging as a powerful tool in the bottom-up fabrication of functional nanostructures for targeted applications. Aromatic π-π stacking plays a significant role by facilitating the persistent supramolecular association of individual subunits to the self-assembled structures of high stability. Understanding, the supramolecular chemistry of the materials interacting through aromatic interactions, is of tremendous interest in not only constructing functional materials but also in revealing the mechanism of molecular assembly in living organisms. This chapter aims to focus on understanding the potential role of π-π interactions in directing and regulating the self-assembly of peptide nanostructures. The scope of the chapter starts with an outline of the history and mechanism of the aromatic π-π interactions. It progresses through the design strategy for the assembly of peptides containing aromatic rings, the conditions affecting the aromatic stacking interactions, their resulting nanoassemblies, properties, and applications. The properties and applications of the supramolecular materials formed through the aromatic stacking interactions are highlighted to provide an increased understanding of the role of weak interactions in the design and construction of novel functional materials.

Electric Field Disruption of Amyloid Aggregation: Potential Noninvasive Therapy for Alzheimer's Disease.

The aggregation of ß-amyloid peptides is a key event in the formative stages of Alzheimer's disease. Promoting folding and inhibiting aggregation was reported as an effective strategy in reducing Aß-elicited toxicity. This study experimentally investigates the influence of the external electric field (EF) and magnetic field (MF) of varying strengths on the in vitro fibrillogenesis of hydrophobic core sequence, Aß16-22, and its parent peptide, Aß1-42. Biophysical methods such as ThT fluorescence, static light scattering, circular dichroism, and infrared spectroscopy suggest that EF has a stabilizing effect on the secondary structure, initiating a conformational switch of Aß16-22 and Aß1-42 from ß to non-ß conformation. This observation was further corroborated by dynamic light scattering and transmission electron microscopic studies. To mimic in vivo conditions, we repeated ThT fluorescence assay with Aß1-42 in human cerebrospinal fluid to verify EF-mediated modulation. The self-seeding of Aß1-42 and cross-seeding with Aß1-40 to verify that the autocatalytic amplification of self-assembly as a result of secondary nucleation also yields comparable results in EF-exposed and unexposed samples. Aß-elicited toxicity of EF-treated samples in two neuroblastoma cell lines (SH-SY5Y and IMR-32) and human embryonic kidney cell line (HEK293) were found to be 15-38% less toxic than the EF untreated ones under identical conditions. Experiments with fluorescent labeled Aß1-42 to correlate reduced cytotoxicity and cell internalization suggest a comparatively smaller uptake of the EF-treated peptides. Our results provide a scientific roadmap for future noninvasive, therapeutic solutions for the treatment of Alzheimer's disease.

Single Crystal Organic Nanoflowers.

Nano-flowers reported so far were mostly constituted of inorganic or hybrid materials. We have synthesized and crystallized a new organic compound, 1, 2-bis(tritylthio)ethane forming an organic nano-flower consisting of single crystalline petals. Crystal structure at nano and micro level indicates that π-π stacking interactions between aromatic systems is the principal factor governing molecular recognition and assembly. Single crystal X-ray Diffraction (S-XRD) supported by Selective Area Electron Diffraction (SAED) experiments indicate the single crystalline nature of the flower-like assembly even at the nanoscale. In order to fabricate the nanoflower as a potential stimulus responsive material; the 'petals' were coated with magnetite nanoparticles, verified by Energy-dispersive X-ray spectroscopic (EDX) analysis. Herein, we have further tested the potential utility of the hybrid material in water remediation as a nano-based adsorbent for removal of heavy metals like chromium.

Modulation of Peptide Based Nano-Assemblies with Electric and Magnetic Fields.

Peptide based nano-assemblies with their self-organizing ability has shown lot of promise due to their high degree of thermal and chemical stability, for biomaterial fabrication. Developing an effective way to control the organization of these structures is important for fabricating application-oriented materials at the molecular level. The present study reports the impact of electric and magnetic field-mediated perturbation of the self-assembly phenomenon, upon the chemical and structural properties of diphenylalanine assembly. Our studies show that, electric field effectively arrests aggregation and self-assembly formation, while the molecule is allowed to anneal in the presence of applied electric fields of varying magnitudes, both AC and DC. The electric field exposure also modulated the morphology of the self-assembled structures without affecting the overall chemical constitution of the material. Our results on the modulatory effect of the electric field are in good agreement with theoretical studies based on molecular dynamics reported earlier on amyloid forming molecular systems. Furthermore, we demonstrate that the self-assemblies formed post electric-field exposure, showed difference in their crystal habit. Modulation of nano-level architecture of peptide based model systems with external stimulus, points to a potentially rewarding strategy to re-work proven nano-materials to expand their application spectrum.

Symmetry-Directed Self-Organization in Peptide Nanoassemblies through Aromatic π-π Interactions.

Almost all biological systems are assemblies of one or more biomolecules from nano- to macrodimensions. Unlike inorganic molecules, peptide systems attune with the conceptual framework of aggregation models when forming nanoassemblies. Three significant recent theoretical models have indicated that nucleation, end-to-end association, and geometry of growth are determined primarily by the size and electrostatics of the individual basic building blocks. In this study, we tested six model systems, differentially modulating the prominence of three design variables, namely, aromatic π-π interactions, local electrostatics, and overall symmetry of the basic building unit. Our results indicate that the crucial design elements in a peptide-based nanoassembly are (a) a stable extended π-π interaction network, (b) size, and (c) overall symmetry of the basic building blocks. The six model systems represent all of the design variables in the best manner possible, considering the complexity of a biomolecule. The results provide important directives in deciding the morphology and crystallinity of peptide nanoassemblies.