Because of their capacity of crossing an intact blood-brain barrier and reaching the brain through an injured barrier or via the nasal epithelium, nanoparticles have been considered as vehicles to deliver drugs and as contrast materials for brain imaging. The potential neurotoxicity of nanoparticles, however, is not fully explored. Using particles with a biologically inert polystyrene core material, we investigated the role of the chemical composition of particle surfaces in the in vitro interaction with different neural cell types. PS NPs within a size-range of 45-70 nm influenced the metabolic activity of cells depending on the cell-type, but caused toxicity only at extremely high particle concentrations. Neurons did not internalize particles, while microglial cells ingested a large amount of carboxylated but almost no PEGylated NPs. PEGylation reduced the protein adsorption, toxicity and cellular uptake of NPs. After storage (shelf-life >6 months), the toxicity and cellular uptake of NPs increased. The altered biological activity of "aged" NPs was due to particle aggregation and due to the adsorption of bioactive compounds on NP surfaces. Aggregation by increasing the size and sedimentation velocity of NPs results in increased cell-targeted NP doses. The ready endotoxin adsorption which cannot be prevented by PEG coating, can render the particles toxic. The age-dependent changes in otherwise harmless NPs could be the important sources for variability in the effects of NPs, and could explain the contradictory data obtained with "identical" NPs.
Engineering of drug nanocarriers combining fine-tuned mucoadhesive/mucopenetrating properties is currently being investigated to ensure more efficient mucosal drug delivery. Aiming to improve the transmucosal delivery of hydrophobic drugs, we designed a novel nanogel produced by the self-assembly of amphiphilic chitosan graft copolymers ionotropically crosslinked with sodium tripolyphosphate. In this work, we synthesized, for the first time, chitosan-g-poly(methyl methacrylate) nanoparticles thiolated by the conjugation of N-acetyl cysteine. First, we confirmed that both non-crosslinked and crosslinked nanoparticles in the 0.05–0.1% w/v concentration range display very good cell compatibility in two cell lines that are relevant to oral delivery, Caco-2 cells that mimic the intestinal epithelium and HT29-MTX cells that are a model of mucin-producing goblet cells. Then, we evaluated the effect of crosslinking, nanoparticle concentration, and thiolation on the permeability in vitro utilizing monolayers of (i) Caco-2 and (ii) Caco-2:HT29-MTX cells (9:1 cell number ratio). Results confirmed that the ability of the nanoparticles to cross Caco-2 monolayer was affected by the crosslinking. In addition, thiolated nanoparticles interact more strongly with mucin, resulting in a decrease of the apparent permeability coefficient (Papp) compared to the pristine nanoparticles. Moreover, for all the nanoparticles, higher concentration resulted in lower Papp, suggesting that the transport pathways can undergo saturation.
Laccases are multi-copper
oxidase enzymes having widespread applications in various biotechnological
fields. However, low stability of free enzymes restricts their industrial
use. Development of effective methods to preserve and even increase
the enzymatic activity is critical to maximize their use, though this
remains a challenge. In the present study we immobilized Trametes versicolor laccase on pH-responsive (and
charge-switchable) Pluronic-stabilized silver nanoparticles (AgNPsTrp). Our results demonstrate that colloidal stabilization
of AgNPsTrp with the amphiphilic copolymer Pluronic F127
enhances enzyme activity (AgNPsTrpF1 + Lac6) by changing the active
site microenvironment, which is confirmed by circular dichroism (CD)
and fluorescence spectroscopy. Detailed kinetic and thermodynamic
studies reveal a facile strategy to improve the protein quality by
lowering the activation energy and expanding the temperature window
for substrate hydrolysis. The immobilized nanocomposite did not show
any change in flow behavior which indirectly suggests that the enzyme
stability is maintained, and the enzyme did not aggregate or unfold
upon immobilization. Finally, assessing the anticancer efficacy of
this nanocomposite in breast cancer MCF-7 cells shows the inhibition
of cell proliferation through β-estradiol degradation and cells
apoptosis. To understand the molecular mechanism involved in this
process, semi qRT-PCR experiments were performed, which indicated
significant decrease in the mRNA levels of anti-apoptotic genes, for
example, BCL-2 and NF-kβ,
and increase in the mRNA level of pro-apoptotic genes like p53 in treated cells, compared to control. Overall, this
study offers a completely new strategy for tailoring nano-bio-interfaces
with improved activity and stability of laccase.
The nose‐to‐brain (N‐to‐B) transport mechanism of nanoparticles through the olfactory epithelium (OE) is not fully understood. Most research utilized nasal epithelial cell models completely deprived of olfactory cells. Aiming to shed light into key cellular pathways, in this work, for the first time, the interaction of polymeric nanoparticles in a 17–483 nm size range and with neutral and negatively and positively charged surfaces with primary olfactory sensory neurons, cortical neurons, and microglia isolated from olfactory bulb (OB), OE, and cortex of newborn rats is investigated. After demonstrating the good cell compatibility of the different nanoparticles, the nanoparticle uptake by confocal laser scanning fluorescence microscopy is monitored. Our findings reveal that neither olfactory nor forebrain neurons internalize nanoparticles. Conversely, it is demonstrated that olfactory and cortical microglia phagocytose the nanoparticles independently of their features. Overall, our findings represent the first unambiguous evidence of the possible involvement of microglia in N‐to‐B nanoparticle transport and the unlikely involvement of neurons. Furthermore, this approach emerges as a completely new experimental tool to screen the biocompatibility, uptake, and transport of nanomaterials by key cellular players of the N‐to‐B pathway in nanosafety and nanotoxicology and nanomedicine.
Nanoneuromedicine investigates nanotechnology to target the brain and treat neurological diseases. In this work, we biofabricated heterocellular spheroids comprising human brain microvascular endothelial cells, brain vascular pericytes and astrocytes combined with primary cortical neurons and microglia isolated from neonate rats. The structure and function are characterized by confocal laser scanning and light sheet fluorescence microscopy, electron microscopy, western blotting, and RNA sequencing. The spheroid bulk is formed by neural cells and microglia and the surface by endothelial cells and they upregulate key structural and functional proteins of the blood-brain barrier. These cellular constructs are utilized to preliminary screen the permeability of polymeric, metallic, and ceramic nanoparticles (NPs). Findings reveal that penetration and distribution patterns depend on the NP type and that microglia would play a key role in this pathway, highlighting the promise of this platform to investigate the interaction of different nanomaterials with the central nervous system in nanomedicine, nanosafety and nanotoxicology.
this work, we designed, characterized, and investigated the
performance of hydrolyzed galactomannan (hGM)-based amphiphilic nanoparticles
for selective intratumoral accumulation in pediatric patient-derived
sarcomas. To create a self-assembly amphiphilic copolymer, the side
chain of hGM was hydrophobized with poly(methyl methacrylate) (PMMA)
by utilizing a graft free radical polymerization reaction. Different
hGM and MMA weight feeding ratios were used to adjust the critical
aggregation concentration and the size and size distribution of the
nanoparticles. The ability to actively target glucose transporter-1
(GLUT-1) was studied by fluorescence confocal microscopy and imaging
flow cytometry in vitro on Rh30 (rhabdomyosarcoma) and patient-derived
Ewing sarcoma (HSJD-ES-001) cell lines with different expression levels
of GLUT-1. Results confirmed that the nanoparticles are internalized
by ∼100% of the cells at 37 °C. Furthermore, we investigated
the biodistribution of the nanoparticles in pediatric patient-derived
models of two deadly musculoskeletal tumors, rhabdomyosarcoma and
Ewing sarcoma. Outstandingly, the intratumoral accumulation of the
nanoparticles correlated very well with the expression level of GLUT1 gene in each patient-derived tumor (P = 0.0141; Pearson’s correlation test). Finally, we demonstrated
the encapsulation capacity of these nanoparticles by loading 7.5%
(w/w) of the hydrophobic first-generation tyrosine kinase inhibitor
imatinib. These findings point out the potential of this new type
of nanoparticle to target GLUT-1-expressing tumors and selectively
deliver anticancer agents.
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