Short Communication

Cellular transport pathways of polymer coated gold nanoparticles

I-Chun Lin, BSc
a
, Mingtao Liang, PhD
a, b
, Tzu-Yu Liu, MSc
a
,
Michael J. Monteiro, PhD
c
, Istvan Toth, PhD
a, b,

a
School of Chemistry and Molecular Bioscience, University of Queensland, Brisbane, Australia
b
School of Pharmacy, University of Queensland, Brisbane, Australia
c
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia
Received 26 July 2011; accepted 25 September 2011
Abstract
The different transport pathways of 5-nm polymer-coated gold nanoparticles (Au NPs) crossing epithelial Caco-2 cell monolayers were
explored. We found that the majority of cationic and neutral Au NPs depended heavily on endocytosis for cellular uptake and transport, and
the anionic charged nanoparticles trafficked preferentially through the tight junctions (i.e., a paracellular pathway). The current study
demonstrates that the surface chemistry of neutral polymer coatings dictate the trafficking through Caco-2 cell monolayers; poly(ethylene
glycol)-coated Au NPs traffic via an endocytosis pathway assisted by microtubules; poly(2,3-hydroxy-propylacrylamide)-coated Au NPs
traffic via endocytosis but assisted by other nonmicrotubular pathways. The Au NPs coated with poly(N-isopropylacrylamide) (hydrophobic
above the lower critical solution temperature of 32C) traffic via either the microtubule-assisted endocytosis pathway or the paracellular
pathway depending on the temperature. This knowledge will aid in the future of the design of nanoparticles as potential oral drug carriers.
From the Clinical Editor: The authors examined different transport pathways of polymer-coated gold nanoparticles to cross epithelial Caco-
2 cells, concluding that surface chemistry of neutral polymer coatings dictates the trafficking through monolayers.
2012 Elsevier Inc. All rights reserved.
Key words: Polymer-coated nanoparticles; Gold nanoparticles; Caco-2 cells; Cellular transport; Endocytosis
Understanding the interaction of polymeric nanoparticles with
intestinal epithelial cells is imperative for their development as
potential oral drug carriers. However, little is known about the
effect of nanoparticle size and surface properties on their cellular
transport behaviors for such systems. We previously studied the
transport of model polymer nanoparticles, made from gold
nanoparticles (Au NPs) coated with a dense polymer shell bearing
cationic, anionic, or neutral (hydrophilic, hydrophobic) side chain
functionalities, across epithelial Caco-2 cells in vitro with sizes
ranging from 5 to 20 nm.
1,2
The smaller and neutral nanoparticles
showed the highest transport efficiency across the cell monolayer,
whereas the larger nanoparticles with hydrophobic or cationic
surface properties showed little or no cellular translocation, being
mostly trapped inside endocytic vesicles within the cells.
In the present study, we sought to unravel the different
mechanisms involved in nanoparticle cellular transport by
investigating the different transport pathways of 5-nm Au NPs
coated with a wide range of polymers (Figure 1) to cross
epithelial Caco-2 cell monolayers. By decreasing the temperature
to 4C, we inhibit the endocytosis pathway and thus differentiate
between endocytosis and paracellular pathways (transport
through tight junctions).
3
If transportation is via the endocytosis
pathway, the addition of colchicine (which irreversibly binds to
tubulin subunits and blocks the microtubular-assisted endocyto-
sis) will further differentiate the type of endocytosis pathway.
4
Microtubules, part of the cytoskeleton, structurally consist of
polymer globular tubulin subunits that form an extensive
network throughout the cell cytoplasm and can help to generate
specific endocytic invaginations at the cell membrane. They have
been shown to be responsible for the intracellular trafficking of
endocytic vesicles across cells.
5
Studying the endocytic
mechanisms of polymer nanoparticles would improve their
design as oral drug carriers.
Methods
The synthesis of polymer-coated 5-nm Au NPs and their
physicochemical characterization are detailed elsewhere.
1,2
The
This work was supported by the Australian Research Council
(DP878733).

Corresponding author: School of Chemistry and Molecular Bioscience,

Australian Institute for Bioengineering and Nanotechnology, The University
of Queensland, Brisbane QLD 4072, Australia.
E-mail address: i.toth@uq.edu.au (I. Toth).
BASIC SCIENCE
Nanomedicine: Nanotechnology, Biology, and Medicine
8 (2012) 811
nanomedjournal.com
1549-9634/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.nano.2011.09.014
Please cite this article as: I.-C. Lin, M. Liang, T.-Y. Liu, M.J. Monteiro, I. Toth, Cellular transport pathways of polymer coated gold nanoparticles.
Nanomedicine: NBM 2012;8:8-11, doi:10.1016/j.nano.2011.09.014
polymers used included the anionic poly(acrylic acid) (PAA),
neutral poly(2,3-hydroxy-propylacrylamide) (PDHA), and cat-
ionic poly(2-aminoethylacrylamide) (PAEA). In addition, a
thermoresponsive poly(N-isopropylacrylamide) (PNIPAM)
polymer (hydrophobic above and hydrophilic below its lower
critical solution temperature of 32C)
6
and poly(ethylene
glycol) (PEG) were also coated onto the 5-nm Au NPs. Details
on materials used and characterization of the polymer-coated Au
NPs are given in Supplementary Materials Sections S1 and S2,
available online at http://www.nanomedjournal.com.
Caco-2 cells (passage 42) were seeded on permeability plates
for approximately 27 days to reach confluency before being used
in the transport assay (Supplementary Materials Sections S3 and
S4). The cellular transport experiments discussed herein were
performed according to established methods.
2
We first evaluated
the transport of the polymer-coated Au NPs (45 g/mL) across
Caco-2 cell monolayers under standard conditions at 37C
(physiological pH, temperature without inhibitors). We then
compared the differences in nanoparticle cellular transport at
4C, and in the absence and presence of the microtubule inhibitor
colchicine (10 M) at 37C. Each experiment was repeated three
times, and nanoparticle concentrations were analyzed by
inductively coupled plasma mass spectrometry.
Results and discussion
The size of the resultant nanoparticles was the same for all
coated Au NPs (5.5 nm), but their charge density changed as
shown in Table 1.
1,2
Following the coating process, these
polymers formed a densely packed shell (0.751.4 chains/nm
2
)
on the Au NP surface, excluding the gold core from undesirable
cellular interactions.
1
From the cellular transport assays (Figure 2), approximately
11% of the neutral PEG- and PDHA-Au NPs transported across to
the basolateral chamber at 37C under physiological conditions.
These results suggested high permeability rates of neutral
nanoparticles in humans when compared to the non permeable
control mannitol (Supplementary Material, Section S5). The
anionic PAA-Au NPs were slightly less permeable, whereas the
hydrophobic PNIPAM- and cationic PAEA-Au NPs displayed
little ability to transport through the Caco-2 cell monolayer, with
Figure 1. The grafting of RAFT polymers and PEG polymer onto 5-nm Au NPs through chemisorption.
9 I.-C. Lin et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 811
high percentage binding to the cells. The trend of permeability was
in agreement with our previous transport assay results.
2
We next
performed the Caco-2 cell assays at 4C to block endocytic
transcellular pathways. A significant decrease (P b 0.05;
Supplementary Materials Section S6) in the transport of neutral
PDHA-, cationic PAEA-, and PEG-Au NPs to the basolateral
chamber of the cell monolayer was observed (Figure 2), indicating
a strong dependence on the endocytosis pathway. Blocking
endocytic uptake will therefore lead to an increased level of
nanoparticles in the top apical layer. Indeed, the amount of PAEA-
Au NPs that remained in the top apical chamber increased from
10% to 30%, further supporting the strong contribution of
endocytosis pathway. Interestingly, we observed only a minor
reduction in the transport of the anionic PAA-Au NPs through cell
monolayer at 4C. This implied that the PAA-Au NPs mainly
utilized the paracellular pathway. To our surprise, the permeability
of the PNIPAM-Au NPs through the cell monolayer increased
substantially at 4C. These PNIPAM-Au NPs were neutral and
hydrophilic at temperatures below their lower critical solution
temperature.
6
One would expect the neutral PNIPAM-Au NPs to
have the same transport behavior as the neutral PEG- and PDHA-
Au NPs at low temperatures because of their similar size and
surface properties. However, unlike the PEG- and PDHA-Au NPs,
which displayed no cellular transport at 4C, PNIPAM-Au NPs
transportation seems to be preferentially via the paracellular
pathway. These results now strongly support that the surface
chemistry plays a more significant role than originally thought in
trafficking neutral nanoparticles.
The addition of colchicine at 37C inhibits endocytosis
assisted by microtubules (Figure 2). There was negligible
transport for neutral PEG-Au NPs and reduced cellular uptake
of the hydrophobic PNIPAM-Au NPs into the cells. The reduced
uptake of PNIPAM-Au NPs resulted in a 2530% increased
accumulation of these nanoparticles in the apical layer when
compared to the standard 37C cell assay, indicating strong
contribution of microtubules for the endocytic uptake and
transport of PNIPAM and PEG-Au NPs under physiological
conditions. Microtubules have long been considered to partici-
pate in various endocytic pathways such as the caveolar-mediated
endocytosis
7
or macropinocytosis.
8
PEG and PNIPAM-Au NPs
most probably utilize these pathways for transcellular transport.
Conversely, colchicine barely affected the cell transport
behavior of other polymer-coated Au NPs (PAA-, PDHA-, and
PAEA-Au NPs). These polymer-coated Au NPs employed other
endocytic pathways through Caco-2 cells, most probably the
clathrin-dependent endocytosis,
9
and other pathways involving
cytoskeletal components such as the actin microfilaments.
10
An
interesting finding from the colchicine study was that we
observed both a microtubule-dependent (PEG-Au NPs) and non-
microtubule-dependent (PDHA-Au NPs) endocytic mechanism
for the transport of neutral nanoparticles across Caco-2 cells.
This observation further supports that the surface chemistry on
the nanoparticles plays a dominant role in the cellular trafficking
of functional nanoparticles.
In summary, the current study demonstrates that the surface
chemistry of neutral polymer coatings dictates the trafficking
through Caco-2 cells, potentially giving high absorption rates in
humans. PEG-Au NPs traffic via an endocytosis pathway
assisted by microtubules, PDHA-Au NPs traffic via endocytosis
but assisted by other (nonmicrotubular) pathways, and PNIPAM-
Au NPs traffic via either a paracellular pathway (hydrophilic) or
microtubule-dependent pathway (hydrophobic) depending on
the temperature. Further studies on nanoparticle transport using
other inhibitors (microfilaments) or examining the involvement
of efflux proteins, could help us in the future to design
nanoparticles that can target specific cellular pathways and
potentially be used as oral drug carriers.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.nano.2011.09.014.
Figure 2. (A) Percentage of nanoparticles remaining in the apical chamber after
3 hours. (B) Percentage of nanoparticles transported to the basolateral chamber
after 3 hours. t-test: n.s., nonsignificant;

P b 0.1;

P b 0.05;

P b 0.01.
Table 1
Surface property data of polymer-coated Au NPs analyzed by DLS (pH 7.4)
PAA-
Au NPs
PDHA-
Au NPs
PAEA-
Au NPs
PNIPAM-
Au NPs
PEG-
Au NPs
Surface charge
(mV)
41.6 3.1 1.3 0.4 22.6 2.4 3.6 0.8 -4.3 1.7
10 I.-C. Lin et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 811
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