4 research outputs found
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
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-bisÂ(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
H‑Ferritin Enriches the Curcumin Uptake and Improves the Therapeutic Efficacy in Triple Negative Breast Cancer Cells
Triple
negative breast cancer (TNBC) is a highly aggressive, invasive,
and metastatic tumor. Although it is reported to be sensitive to cytotoxic
chemotherapeutics, frequent relapse and chemoresistance often result
in treatment failure. In this study, we developed a biomimetic nanodrug
consisting of a self-assembling variant (HFn) of human apoferritin
loaded with curcumin. HFn nanocage improved the solubility, chemical
stability, and bioavailability of curcumin, allowing us to reliably
carry out several experiments in the attempt to establish the potential
of this molecule as a therapeutic agent and elucidate the mechanism
of action in TNBC. HFn biopolymer was designed to bind selectively
to the TfR1 receptor overexpressed in TNBC cells. HFn-curcumin (CFn)
proved to be more effective in viability assays compared to the drug
alone using MDA-MB-468 and MDA-MB-231 cell lines, representative of
basal and claudin-low TNBC subtypes, respectively. Cellular uptake
of CFn was demonstrated by flow cytometry and label-free confocal
Raman imaging. CFn could act as a chemosensitizer enhancing the cytotoxic
effect of doxorubicin by interfering with the activity of multidrug
resistance transporters. In addition, CFn exhibited different cell
cycle effects on these two TNBC cell lines, blocking MDA-MB-231 in
G0/G1 phase, whereas MDA-MB-468 accumulated in G2/M phase. CFn was
able to inhibit the Akt phosphorylation, suggesting that the effect
on the proliferation and cell cycle involved the alteration of PI3K/Akt
pathway
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