Detection and Characterization of Different Brain-Derived Subpopulations of Plasma Exosomes by Surface Plasmon Resonance Imaging

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases

Fe₃O₄ nanoparticles (MNP, a) synthesized in organic solvent and transferred to a water solution using PMA amphiphilic polymer (PMNP, b).

<p>MNP and PMNP were highly monodisperse in size as it is shown by TEM images (scale bars = 40 nm,). Part of the highly concentrated PMNP suspension (8 mg mL^–1) was incorporated in a w/o cream (0.8 wt % concentration) (c).</p

Cytofluorimetric analysis showing PMNP nanoparticles uptake by mouse skin and lymph node cells.

<p>PMNP suspension (a, upper panels). Skin CD45-positive and negative cells showing CFSE incorporation. Note that most of the skin cells uptake PMNP nanoparticles administered with the cream formulation. (a, lower panels) CFSE-positive cells in the lymph nodes of mice that received PMNP nanoparticles via cream formulation or via sc administration. Note that only with sc PMNP administration, nanoparticle-positive cells can be detected in the draining lymph nodes. (b) Lymph node macrophages and dendritic cells, identified as CD11b- and CD11c-positive cells respectively, showing CFSE incorporation. Note that only when PMNP are administered sc, CFSE positive macrophages and dendritic cells can be detected in the lymph nodes.</p

Histological microphotograph of normal human skin section.

<p>Haematoxylin and eosin staining (original magnification 40×) (a). <i>In vitro</i> diffusion studies of PMNP colloidal suspension or cream in human skin were carried out using Franz diffusion cells and diffused PMNP were quantified by ICP-OES analysis (b).</p

Fates of nanoparticles depending on the route of skin administration.

<p>Nanoparticle administered in a cream formulation are taken up by all the skin cell types and do no reach the draining lymph nodes. Nanoparticle administered with a sc injection in aqueous suspension are efficiently transported to the draining lymph nodes.</p

Antiproliferative Effect of ASC-J9 Delivered by PLGA Nanoparticles against Estrogen-Dependent Breast Cancer Cells

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with “multidrug” resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis­(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the <i>in vitro</i> drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed <i>ad hoc</i>. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer

Polymer Nanopillar–Gold Arrays as Surface-Enhanced Raman Spectroscopy Substrate for the Simultaneous Detection of Multiple Genes

In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-enhanced Raman spectroscopy (SERS) application and tested in a biochemical assay for the simultaneous detection of multiple genetic leukemia biomarkers. The special fabrication process combining soft lithography and plasma deposition techniques allows tailoring of the structural and chemical parameters of the crystal surfaces. In this way, it has been possible to tune the plasmonic resonance spectral position close to the excitation wavelength of the monochromatic laser light source in order to maximize the enhancing properties of the substrate. Samples are characterized by scanning electron microscopy and reflectance measurements and tested for SERS activity using malachite green. Besides, as the developed substrate had been prepared on a simple glass slide, SERS detection from the support side is also demonstrated. The optimized substrate is functionalized with thiol-modified capture oligonucleotides, and concentration-dependent signal of the target nucleotide is detected in a sandwich assay with labeled gold nanoparticles. Gold nanoparticles functionalized with different DNA and various Raman reporters are applied in a microarray-based assay recognizing a disease biomarker (Wilms tumor gene) and housekeeping gene expressions in the same time on spatially separated microspots. The multiplexing performance of the SERS-based bioassay is illustrated by distinguishing Raman dyes based on their complex spectral fingerprints