مجال
التميز
|
تميز دراسي وبحثي
|
|
|
البحوث المنشورة
|
|
البحث (1):
|
|
عنوان البحث:
|
Mechanisms
of Nanoparticle Internalization and Transport Across an Intestinal Epithelial
Cell Model: Effect of Size and Surface Charge
|
رابط إلى البحث:
|
Click here
|
تاريخ النشر:
|
19 October
2014
|
موجز عن البحث:
|
This
study investigated the effect of nanoparticle size (50 and 100 nm) and
surface charge on their interaction with Caco-2 monolayers as a model of the
intestinal epithelium, including cell internalization pathways and the level
of transepithelial transport. Initially, toxicity assays showed that cell
viability and cell membrane integrity were dependent on the surface charge
and applied mass, number, and total surface area of nanoparticles, as tested
in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This
also identified suitable nanoparticle concentrations for subsequent cell
uptake experiments. Nanoparticle application at doses below half maximal
effective concentration (EC50) revealed that the transport efficiency (ratio
of transport to cell uptake) across Caco-2 cell monolayers is significantly
higher for negatively charged nanoparticles compared to their positively
charged counterparts (of similar size), despite the higher level of
internalization of positively charged systems. Cell internalization pathways
were hence probed using a panel of pharmacological inhibitors aiming to
establish whether the discrepancy in transport efficiency is due to different
uptake and transport pathways. Vesicular trans-monolayer transport for both
positively and negatively charged nanoparticles was confirmed via inhibition
of dynamin (by dynasore) and microtubule network (via nocodazole), which
significantly reduced the transport of both nanoparticle systems. For
positively charged nanoparticles a significant decrease in internalization
and transport (46% and 37%, respectively) occurred in the presence of a
clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition
(42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride), and under cholesterol
depletion (38%; via methyl-β-cyclodextrin), but remained unaffected by the
inhibition of lipid raft associated uptake (caveolae) by genistein. On the
contrary, the most prominent reduction in internalization and transport of
negatively charged nanoparticles (51% and 48%, respectively) followed the
inhibition of lipid raft-associated pathway (caveolae inhibition by
genistein) but was not significantly affected by the inhibition of clathrin
pathway.
Keywords: Cell uptake; Caco-2; endocytosis;
epithelial cells; nanoparticles; nanoparticle transport; nanotoxicity
|
|
|
المؤتمرات العلمية:
|
|
المؤتمر (1):
|
|
عنوان المؤتمر:
|
UKICRS conference, April 2012
|
تاريخ الإنعقاد:
|
May 2012
|
مكان
الإنعقاد:
|
Birmingham, UK
|
طبيعة المشاركة:
|
Poster presentation
|
عنوان المشاركة:
|
Interaction of nanoparticles with
epithelial cells: particle properties, influencing toxicity, cell uptake and
transport
|
ملخص المشاركة:
|
The
interaction of nanoparticles with cells has been under extensive interest
during recent years because they can play a vital role in gene and drug
delivery. The work we present involves the interaction of nanoparticles with
epithelial cells (Caco-2 cells). In vitro experiments are focused on the
physicochemical properties of nanoparticles, cytotoxicity and several factors
such as duration of incubation, particle size, particle concentration,
chemical surface properties and temperature and how these factors can
influence cellular uptake and transport. Different sizes and concentrations
of aminated polystyrene nanoparticles (50, 100, 200 nm) were used in this
study. The size and zeta potential of nanoparticles in biological buffer were
determined using dynamic light scattering and zetasizing. Toxicity studies of
50 and 100 nm nanoparticles were also investigated by using MTS assay (can
measure metabolic function), LDH assay (measure the membrane integrity as a
function of the amount of cytopasmic LDH released into the medium) and TEER.
Higher concentrations of 50 and 100 nm aminated polystyrene nanoparticles can
induce higher cellular toxicity when compared to lower concentrations.
Uptake
and transport polystyrene nanoparticles (50, 100, 200 nm; aminated nanoparticles
{positively charged} and 100 nm carboxylated nanoparticles {negatively
charged}) across Caco-2 cells was also investigated. This work found that
cellular uptake and transport was increased by increasing the concentration
of nanoparticles (100 µg/ml > 50 µg/ml > 25 µg/ml), decreasing the
diameter of aminated nanoparticles (50 nm > 100 nm > 200 nm) and
increasing the temperature to 37°C. In this study we found the uptake of
nanoparticles was decreased (60%) at 4°C which suggests that the uptake of these
particles through Caco-2 cells can be energy dependent endocytic process.
Furthermore, uptake and transport studies across Caco-2 cells were also
conducted to investigate the effect of two different functional groups at the
surface of polystyrene nanoparticles. Uptake and transport of 100 nm aminated
and carboxylated polystyrene nanoparticles during 4 hours (100 µg/ml) was
compared and we found that carboxylated nanoparticles experience a higher
degree of repulsion from the negative cell membrane and can have a higher
transport across the cells compared to aminated nanoparticles. This may be
due to aminated nanoparticles (positively charged) becoming entrapped at the
cell surface and undergo higher uptake, rather than transport across the
cells than negatively charged particles. We propose this occurs through
electrostatic interactions.
These
findings create a field which explores surface modifications of nanoparticles
and how these can be used to assess new formulations for oral administration
of therapeutic proteins or peptides.
|
|
|
المؤتمر (2):
|
|
عنوان المؤتمر:
|
Nanomedicine conference
|
تاريخ الإنعقاد:
|
26-27 March
2014
|
مكان
الإنعقاد:
|
Edinburgh
|
طبيعة المشاركة:
|
Poster presentation
|
عنوان المشاركة:
|
Trafficking pathways of oppositely charged nanoparticles
in intestinal Caco-2 monolayers.
|
ملخص المشاركة:
|
Endocytosis
can be defined as the internalization of the plasma membrane, which includes
concomitant engulfment of extracellular fluid and extracellular material1.
Mechanisms by which nanoparticles internalise and transport into/across
intestinal epithelial cells (polarised cells) are poorly defined. Endocytic
mechanisms of cellular uptake and transcellular transport of nanoparticles
can be probed using pharmacological inhibitors2. This study aims to determine
whether negatively and positively charged nanoparticles show different uptake
and transport mechanisms in differentiated Caco-2 (human colon carcinoma)
monolayers as an intestinal epithelial model.
Work found that clathrin pathway (identified
from its sensitivity to chlorpromazine inhibitor) participates in the
internalisation and transport of positively charged nanoparticles, as these
processes decreased by 46% and 37%, respectively. In the case of negatively
charged nanoparticles inhibition of the clathrin pathway had no significant
effect. Genistein (inhibitor of lipid raft-associated receptors) exhibited an
effect on the internalisation and transport of negatively charged
nanoparticles, as suggested by inhibition of these phenomena by 51% (cell
internalisation) and 48%, (transport).
Data also suggest that macropinocytosis and cholesterol depletion are
involved in the uptake and transport of both positively and negatively
charged nanoparticles. Microtubule network also participates in the transport
of positively and negatively charged nanoparticles. This work is therefore
important in the field of drug delivery of biotherapeutics, where
nanoparticle carriers have been suggested as drug carriers that could
potentially cross the mucosal barrier.
|
|
|
المؤتمر (3):
|
|
عنوان المؤتمر:
|
Saudi scientific international conference
|
تاريخ الإنعقاد:
|
1-2
February 2014
|
مكان
الإنعقاد:
|
Edinburgh
|
طبيعة المشاركة:
|
Poster presentation
|
عنوان المشاركة:
|
Effect of nanoparticle surface charge on
interaction with epithelial cells.
|
ملخص المشاركة:
|
The interaction of nanoparticulates
designed for drug delivery (eg polymeric nanoparticles, liposomes or
emulsions) with cells is governed by the nature of their size, shape and
surface characteristics. The surface properties, including charge, surface
curvature, and surface hydrophilicity/hydrophobicity have extensively been
exploited with the aim to provide opportunities for site-specific drug
delivery.
The
aim of the present work is to assess the effect of nanoparticle surface
charge on their interaction with cells in an established in vitro intestinal
epithelium model based on Caco-2 (human colon carcinoma) cells cultured on
permeable membranes. This model is capable of forming cell layers of
polarized, differentiated cells with intercellular tight junction structures,
presenting a physical and biochemical barrier to the passage of ions and
molecules. Toxicity of nanoparticles would vary with their physicohemical
parameters. The dependence of cytotoxicity on particle size and surface
charge of nanoparticles was investigated in this study. Two different sizes
(50 nm and 100 nm) and two different surface charges (amine and carboxyl
groups) of nanoparticles were tested with MTS assay (to assess the metabolic
activity) and LDH assay (to assess cell membrane integrity). Nanoparticles
with positive charge have a higher toxicity than those of with negative
charge. Nanoparticles was concentration and size dependent toxicity in terms
of mass concentration, number of particles and the total surface area.
Nanoparticles of the larger size are less toxic than those of the smaller
size. However, in terms of number of nanoparticles and total of surface area,
the larger size of nanoparticles more toxic than those of the smaller size.
This work found that Cellular uptake and transport of aminated and
carboxylated nanoparticles was concentration, size and temperature dependent.
Cellular uptake and transport was increased by increasing the concentration
of nanoparticles, decreasing the diameter of nanoparticles and increasing the
temperature to 37°C. We investigated that Cellular uptake and transport of
carboxylated nanoparticles exhibit a higher degree of repulsion from the
negative cell membrane and can have a higher transport across the cells
compared to aminated nanoparticles. This may be due to aminated nanoparticles
(positively charged) becoming entrapped at the cell surface and undergo
higher uptake, rather than transport across the cells than negatively charged
particles.
These findings create a field which
explores surface modifications of nanoparticles and how these can be used to
assess new formulations for oral administration of therapeutic proteins or
peptides.
|
|
|
المؤتمر (4):
|
|
عنوان المؤتمر:
|
Nanoparticle-based
technologies for cell tracking conference
|
تاريخ الإنعقاد:
|
1-2 July 2013
|
مكان
الإنعقاد:
|
Liverpool,
UK
|
طبيعة المشاركة:
|
Poster
Presentation
|
عنوان المشاركة:
|
Trafficking
pathways of oppositely charged nanoparticles in intestinal Caco-2 monolayers.
|
ملخص المشاركة:
|
Endocytosis
can be defined as the internalization of the plasma membrane, which includes
concomitant engulfment of extracellular fluid and extracellular material1.
Mechanisms by which nanoparticles internalise and transport into/across
intestinal epithelial cells (polarised cells) are poorly defined. Endocytic
mechanisms of cellular uptake and transcellular transport of nanoparticles
can be probed using pharmacological inhibitors2. This study aims to determine
whether negatively and positively charged nanoparticles show different uptake
and transport mechanisms in differentiated Caco-2 (human colon carcinoma)
monolayers as an intestinal epithelial model.
Work
found that clathrin pathway (identified from its sensitivity to
chlorpromazine inhibitor) participates in the internalisation and transport
of positively charged nanoparticles, as these processes decreased by 46% and
37%, respectively. In the case of negatively charged nanoparticles inhibition
of the clathrin pathway had no significant effect. Genistein (inhibitor of
lipid raft-associated receptors) exhibited an effect on the internalisation
and transport of negatively charged nanoparticles, as suggested by inhibition
of these phenomena by 51% (cell internalisation) and 48%, (transport). Data also suggest that macropinocytosis and
cholesterol depletion are involved in the uptake and transport of both
positively and negatively charged nanoparticles. Microtubule network also
participates in the transport of positively and negatively charged
nanoparticles. This work is therefore important in the field of drug delivery
of biotherapeutics, where nanoparticle carriers have been suggested as drug
carriers that could potentially cross the mucosal barrier.
|