Artificial polymeric receptors on the cell surface promote the efficient cellular uptake of quantum dots

Article abstract of DOI:10.1039/c1ob05420a

In our previous paper, secondary-amine appended cationic polymer 1 was used as a scaffold to display artificial receptors on a cell surface (R. Kamitani et al., ChemBioChem, 2009, 10, 230). This polymer can be retained on the cell surface for more than 30

Full text of DOI:10.1039/c1ob05420a

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PAPER
Artificial polymeric receptors on the cell surface promote the efficient cellular
uptake of quantum dots†
Kenichi Niikura,*a Katsuyuki Nambara,b Takaharu Okajima,c Ryosuke Kamitani,b Shin Aoki,d
Yasutaka Matsuoa and Kuniharu Ijiro*a
Received 17th March 2011, Accepted 18th May 2011
DOI: 10.1039/c1ob05420a
In our previous paper, secondary-amine appended cationic polymer 1 was used as a scaffold to display
artificial receptors on a cell surface (R. Kamitani et al., ChemBioChem, 2009, 10, 230). This polymer
can be retained on the cell surface for more than 30 min before being slowly internalized into the cells.
In this study, our aim is to achieve the efficient internalization of quantum dots (QDs) into target cells
via artificial receptors on the polymer. As a receptor molecule, N-acetylglucosamine (GlcNAc) moieties
were introduced into the polymer, and GlcNAc binding protein-displaying QDs were used as a ligand.
We found that ligand-presenting QDs could be internalized effectively into cells via polymer-mediated
endocytosis, whereas QDs were not internalized into untreated cells. These data suggest that our
method based on cell-surface engineering using polymers affords a new approach to the delivery of
various poorly permeable nanoparticles into cells.
Instead of natural receptors, the display of artificial receptors
on the cell surface offers a valuable approach to inducing cellular
Introduction
uptake of ligand molecules.17–21 An example of the noncovalent
display of receptor molecules on the cell membrane can be found
in the work of Peterson and co-workers, who have shown that
cholesterol derivatives linked to the binding motif for proteins
and drugs act as artificial receptors for efficient drug delivery.18
These bound molecules are reported to be internalized into cells,
and the cholesterol derivatives are returned to the cell membrane
within a relatively short period. These cell-surface engineering
techniques will increase the number of potential ligand–receptor
combinations and make the molecular design of a variety of
nanoparticle surfaces possible.
We have reported that the presence of a secondary amine allows
polymers to be retained on the cell surface for approximately
30 min before they are slowly internalized into cells.1 Our aim
in this study is to exploit these amine polymers as a scaffold
to display artificial receptors on the cell surface, and facilitate
intracellular delivery of the ligand-presenting QDs via artificial
receptor–ligand interactions on the plasma membrane (Fig. 1).
Although the polymer coating of cells has been reported by several
groups,21,22 there have been few reports showing that the polymer
coating on cells enhances the cellular uptake of nanoparticles.
This method has an advantage in that a large number of receptors
can be accumulated on the cell surface. The specific binding
of ligand molecules to receptors on the polymer is expected to
induce the subsequent cellular uptake of the ligands. The receptors
clustered along the polymer chain provide multivalent interactions
for ligand molecules on the cell membrane, thereby effectively
inducing endocytosis of nanoparticles.
The development of a method for nanoparticle delivery into
mammalian cells is of great interest both for the basic insights
into cellular function that it can provide, and for its potential
applications in biomedicine and diagnostics.2,3 In particular,
quantum dots (QDs) have gained much interest in the past decade
for in vitro and in vivo imaging due to their high quantum
yield, size-dependent emission spectra, and resistance to chemical
degradation.4–8 The internalization of QDs into cells is the first
and essential step for the labeling of cellular components. In
order to accelerate the uptake of nanoparticles by target cells, the
nanoparticle surface has been chemically modified with a variety
of ligands, recognized by target cells, to induce receptor-mediated
endocytosis.9–12 However, this strategy of nanoparticle delivery is
useful only when the target cells overexpress known receptors.
Although modification of cationic peptides and polymers with
QDs to gain more efficient internalization has been widely
explored,13–16 some problems remain, such as aggregation of QDs
and their toxicity.
aResearch Institute for Electronic Science (RIES), Hokkaido, University,
Kita21, Nishi10, Kita-Ku, Sapporo, 001-0021, Japan. E-mail: kniikura@
poly.es.hokudai.ac.jp
bDivision of Chemistry, Graduate School of Science, Hokkaido University,
Japan
cGraduate School of Information Science and Technology, Hokkaido Uni-
versity, Japan
dFaculty of Pharmaceutical Sciences, Tokyo University of Science, 2641,
Yamazaki, Noda, 278-8510, Japan
† Electronic supplementary information (ESI) available: Synthetic proce-
dures and identification of compounds. See DOI: 10.1039/c1ob05420a
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Fig. 1 Schematic presentation of QD internalization mediated by artificial receptor-appended amine polymers.
In our previous paper,1 we compared retention times on the
cell surface of primary- and secondary-amine appended polymers.
Herein, we synthesized secondary-, tertiary- and quaternary-
amine appended polymers (referred to as s-, t- and q-amine
polymers, respectively), and compared the intracellular behavior
of these polymers (Scheme 1). GlcNAc, as an artificial receptor,
was conjugated to the s-amine polymer (referred as GlcNAc
polymer), and displayed on the cell surface along the polymer. Two
different types of cells, human fibrosarcoma cell line HT1080 cells
and human fibroblast cell line TIG1-20 cells, were used to assess
nanoparticle delivery by receptor-appended polymer mediation.
We demonstrate that QDs coated with GlcNAc-binding protein
or wheat germ agglutinin (WGA) as ligand molecules can be taken
into HT1080 cells through specific binding to GlcNAc moieties
and subsequent artificial receptor-mediated endocytosis.
sized by the modification of poly(glycidylmethacrylate) (PGMA;
Mn = ca. 25 600, Mw = ca. 33 000, Mw/Mn = 1.29). Conjuga-
tion of PGMA with ethanolamine and dimethylamine gave the
secondary- and tertiary-amine polymers, respectively (Scheme 1).
The construction of quaternary ammonium was carried out by the
addition of an equimolecular amount of trimethylamine and HCl,
according to the previously reported method.23 For visualization
in cells, a small fraction (molecular ratio: 1%) of the epoxide
group was simultaneously reacted with amine-derived fluorescein
to afford a fluorescent probe to monitor their localization in cells,
and with amine-introduced GlcNAc (molecular ratio: 20% to total
epoxide groups) to generate GlcNAc polymer 6.
In this paper, we compare retention times on the cell surface
of Fluorescein isothiocyanate isomer I (FITC) labeled s-amine
polymer 1, t-amine polymer 2 and q-amine polymer 3. These s-, t-
and q-amine polymers were incubated separately with HeLa cells
for 10 min and, after rinsing with PBS buffer, their localizations
were observed using confocal laser scanning microscopy (CLSM)
(Fig. 2A). The q-amine polymer 3 was rapidly internalized into
cells. In contrast, both polymers 1 and 2 were still localized on
Scheme 1 Chemical structures of various amine-appended polymers 1–5
(1, 4: s-amine polymer, 2, 5: t-amine polymer and 3: q-amine polymer) and
GlcNAc polymer 6. Based on Mw value, average polymerization degree (n)
of PGMA was calculated as 186.
Results and discussion
A. Differences in cellular uptake between secondary-, tertiary-
and quaternary-amine polymers
Fig. 2 (A) Fluorescence and differential interference contrast (DIC)
images of HeLa cells after incubation with polymers 1–3 for 10 min.
The nuclei were stained with Hoechst 33342. (B) pH titration curves of
polymers 4, 5 and PAA at 25 ◦C with I = 0.1 (NaNO3).
Synthetic procedures and identification of compounds are de-
scribed in the ESI†. The amine-appended polymers were synthe-
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the cell surface after incubation for 10 min. Since these polymers
were highly water soluble, all cells uniformly interacted with the
polymers. Similar results for membrane localization were also
obtained in NIH3T3 and Jurkat cells, indicating a general trend
in polymer retention (Fig. S1†).
After incubation for 10 min, we investigated the cellular uptake
of polymers 1–3 by flow cytometry (Fig. S2†). The q-amine
polymer 3 was the most readily incorporated into cells among
the three polymers, followed by the s-amine polymer 1. This result
is consistent with that obtained from CLMS imaging, in which the
q-amine polymer 3 was seen to be well-internalized into the cells.
This indicates that q-amine polymer 3 is not suitable for displaying
artificial receptors on the plasma membrane because it is rapidly
internalized into the cells. From these results, we concluded that
s-amine polymer 1 would be the most effective cationic polymer
for the display of artificial receptors on the cell surface.
We speculated that the differences in the degree of protonation
between polymers would be a dominant factor in determining
cell-membrane retention. The protonation behavior of the amine
moieties in the s- and t-amine polymers 4, 5 and primary amine
polymer, poly(allylamine) (PAA), as a control, were studied by
potentiometric pH titration. The deprotonation process of each
hydrochloride-formed polymer was monitored by titration with
NaOH. The raw titration data were transformed to give the
degree of protonation H as a function of pH using the BEST
program24,25 (Fig. 2B), and ionization degrees at pH 7.4 are
summarized in Table 1. In the case of PAA, the degree of
protonation was found to be 0.91 at pH 7.4, which is in good
agreement with the results shown in previous reports.26 On the
other hand, the s- and t-amine polymers 4 and 5 showed much
lower ionization degrees of 0.19 and 0.28 at pH 7.4, respectively.
Smits et al. reported that the protonation of linear polycations
did not proceed completely in an experimentally accessible pH
range.27 This could be due to the electrostatic suppression of the
polymer amine groups by neighboring amine cations via through-
space interactions. The cationic polymers are bound to negatively-
charged heparan sulfates on the extracellular domain of syndecans
of the cell membrane though electrostatic binding, and this induces
syndecan-assembly, thus leading to endocytosis.28 Since s- and t-
amine polymers 4 and 5 have a lower density of cationic residues
than that of PAA and q-amine polymer at a neutral pH, the
weaker electrostatic interactions between the plasma membrane
and polymers would suppress rapid cellular uptake.
Fig. 3 Fluorescence images of HT1080 and TIG1-20 cells (A) after
incubation with s-amine polymer 1 for 10 min, (B) after incubation with
polymer 1 for 10 min, washed with PBS buffer and subsequent culture
for 1 h in serum-free medium at 37 ◦C. Green; fluorescence (FITC) from
polymer 1, Blue; Hoechst 33342.
HT1080 cells within 1 h, whereas it was retained on the plasma
membrane of TIG1-20 cells even after 1 h (Fig. 3B). These findings
are attributable to differences in endocytic activity between these
two cell lines. In order to identify the path of internalization,
we examined the effect of temperature on the cellular uptake of
polymer 1. The uptake of polymer 1 was drastically inhibited at
4
◦C and, it remained localized on the plasma membrane (Fig.
B. Localization of the s-amine polymer 1 on HT1080 and
4). This result demonstrates that endocytosis is the major path of
TIG1-20 cells
s-amine polymer 1 into HT1080 cells.
We compared the internalization behavior of the s-amine polymer
1 using two kinds of cells, HT1080 cells and TIG1-20 cells. In
both cells, polymer 1 was localized on the plasma membrane for
10 min (Fig. 3A). However, polymer 1 was internalized into the
C. GlcNAc-mediated cellular uptake of WGA-presenting
quantum dots
As a receptor molecule, we appended GlcNAc moieties onto the
polymer side chains. We predicted that the GlcNAc moieties of
GlcNAc polymer 6 would act as artificial receptors on the plasma
membrane as few terminal GlcNAc moieties were present. Similar
to the results obtained for polymer 1, GlcNAc polymer 6 was
localized on the plasma membrane of both cells for 10 min and was
then slowly internalized into HT1080 cells within 1 h, whereas it
was retained on the plasma membrane of TIG1-20 cells even after
1 h (Fig. S3†). These results indicate endocytosis-active cells can
Table 1 The ionization degree of s-amine polymer 4, t-amine polymer 5
and PAA at pH 7.4
Polymer
Ionization degree at pH 7.4
4
0.19
0.28
0.91
5
PAA
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Fig. 4 Fluorescence images of HT1080 cells incubated with polymer 1
for 10 min, washed with PBS buffer and subsequent culture for 1 h in
serum-free medium at 4 ◦C.
Fig. 6 Flow cytometry analysis of HT1080 and TIG1-20 cells after
incubation with WGA-QDs. Cells were pretreated with polymer 4 or 6.
In this experiment, polymer 6 without the appended FITC was used to
detect fluorescence from QDs only.
easily internalize ligand molecules through the use of polymeric
artificial receptors. The total amount of GlcNAc polymer in the
HT1080 cells was quantified by flow cytometry analysis. The
fluorescence intensity of GlcNAc polymer 6 after incubation for
1 h was approximately 70% of that after just 10 min incubation
(Fig. S4†). This result shows that most of the plasma membrane-
retained polymers did not dissociate from the plasma membrane
but were internalized into the cells.
After adding GlcNAc polymer 6 to cells, the internalization
of WGA protein-coated quantum dots (WGA-QDs) through
GlcNAc–WGA interactions was monitored by CLSM (Fig. 5).
HT1080 and TIG1-20 cells were incubated with GlcNAc polymer
6 for 10 min. After washing the cells with a buffered solution,
WGA-QDs were added to the media and incubated for 1 h. In
the HT1080 cells, fluorescence from the WGA-QDs was detected
inside the cells (Fig. 5A), whereas the WGA-QDs were localized
on the cell surface of the TIG1-20 cells (Fig. 5B). As a control,
WGA-QDs incubated in the absence of GlcNAc polymer 6 were
not internalized into the HT1080 or TIG1-20 cells even after
incubation for 1 h (Fig. S5†). These CLSM data were also
supported by the results of flow cytometry analysis (Fig. 6). For
both HT1080 and TIG1-20 cells, the fluorescence from QDs can be
detected only after the treatment of cells with GlcNAc polymers,
but not polymer 4.
Importantly, it should be noted that the premixture of WGA-
QDs and GlcNAc polymer under the same conditions, did not
result in cellular uptake (Fig. S6†). This means that the sequential
addition of the polymer and WGA-QDs is essential for efficient
QD uptakes. Furthermore, the addition of s-amine polymer 1
instead of GlcNAc polymer 6 did not result in any significant
cellular uptake of WGA-QDs (Fig. S7†), and the addition of an
excess of GlcNAc (10 mM) in medium also did not result in any
significant cellular uptake of WGA-QDs (Fig. S8†). These data
indicate that WGA-QDs were selectively taken into HT1080 cells
via the plasma membrane-retained polymers displaying artificial
GlcNAc receptors, but not nonspecific endocytosis.
Conclusions
In this study, we concluded that the low cationic density of the
secondary-amine appended polymers is a key prerequisite for long-
term retention on the cell membrane. Monosaccharide GlcNAc
moieties were introduced into the polymer as an artificial receptor.
The GlcNAc moieties were found to work as a receptor for the
Fig. 5 Fluorescence images of (A) HT1080 and (B) TIG1-20 cells treated with polymer 6 for 10 min and then incubated with WGA-QDs for 1h.
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binding and subsequent internalization of ligand-presenting QDs
via the endocytotic pathway. In contrast, preconjugation of WGA-
QDs with the GlcNAc-polymer did not induce the uptake of QDs
into cells, indicating that the sequential addition of the polymer
and QDs is critical for this approach. Particularly, our approach is
useful for cells with high endocytotic activity. The application
of this technique would expand the usefulness of cell-surface
engineering using artificial ligand–receptor interactions towards
cell-specific delivery of nanoparticles into cells.
Incubation of cells with each polymer for CLSM
HeLa (1.0 ¥ 105) cells, HT1080 (1.25 ¥ 105) cells and TIG1-20
(2.0 ¥ 105) cells were seeded in 35 mm glass base dishes separately
and cultured for 1 day in the above-mentioned conditions. The
cells were washed with PBS (2 ¥ 1.0 mL), then Opti-MEM
(2.0 mL) and Hoechst 33342 (2.0 mL) were added. After incubation
at 37 ◦C for 3 min, the cells were washed with PBS (2 ¥ 1.0 mL),
and then each polymer, dissolved in Opti-MEM to a compound
concentration of 25 mg mL-1, was added to the cells. The cells were
incubated in the presence of the polymer at 37 ◦C for 10 min,
washed with PBS (2 ¥ 1.0 mL), and the fluorescence was measured
by CLSM.
Experimental
General remarks
Inhibition of endocytosis of polymer 1
Synthetic procedures for the polymers are described in the supple-
mentary material (ESI†). Gel permeation chromatography (GPC)
analysis was carried out at 40 ◦C on the HLC-8220 GPC system
(TOSOH, Japan) equipped with a TSK gel Super HM-M column
(TOSOH, Japan). Chloroform was used as an eluent at a flow rate
of 0.3 mL min-1. Polystyrene standards (Polymer Laboratories,
USA) were used to calibrate the GPC system. Confocal laser
scanning microscopy (CLSM) employed an Olympus FV300
microscope. Hoechst 33342 was excited with a 405 nm argon ion
laser and emitted photons were collected through a 445/15 nm
band pass filter. Fluorescein isothiocyanate isomer I (FITC) was
excited with a 488 nm argon ion laser and emitted photons
were collected through a 510 nm long pass filter. WGA-QDs
(Invitrogen, Q12021MP, USA) were excited with a 405 nm laser
and emitted photons were collected through a 610 nm long pass
filter. All images were processed with Adobe Photoshop 7.0.
HT1080 (1.25 ¥ 105) cells were seeded in 35 mm glass base dishes
and cultured for 1 day in the above-mentioned conditions. The
cells were washed with PBS (2 ¥ 1.0 mL), and s-amine polymer 1
dissolved in Opti-MEM was added to the cells. After incubation
at 37 ◦C for 10 min, the cells w◦ere washed with PBS (2 ¥ 1.0 mL).
The cells were incubated at 4 C for 1 h, washed with PBS (2 ¥
1.0 mL), then Opti-MEM (2.0 mL) and Hoechst 33342 (2.0 mL)
were added. After washing with PBS (2 ¥ 1.0 mL), the fluorescence
was measured by CLSM.
Flow cytometry analysis of each polymer-treated cells
Cells treated with each polymer prepared by the above mentioned
method were treated with Trypsin/EDTA (0.05%) at 37 ◦C for
3 min. After centrifugation, the cells were resuspended in PBS
(500 mL) and analyzed by the flow cytometer.
Potentiometric pH titration
Cellular uptakes of WGA-QDs through GlcNAc polymer 6
pH values were determined with a potentiometric automatic
titrator (AT-420, KEM, Japan). The electrode system was cal-
ibrated with pH 4.01, 6.86, 9.18 buffer solutions and checked
by the duplicate theoretical titration curves of 0.10 M HCl aq.
solution with 0.10 M NaOH aq. solution at 25 ◦C and ionic
strength (I) = 0.10 M (NaNO3) in high- and low-pH regions.
Aqueous solutions (50 mL) of polymers (1.00 mM; amine moiety
concentration) with 1.0 equivalent HCl adjusted to 0.10 M with
NaNO3 was titrated with 0.1 N NaOH aq. The temperature was
HT1080 (1.25 ¥ 105) cells and TIG1-20 (2.0 ¥ 105) cells were seeded
in 35 mm glass base dishes separately and cultured for 1 day in the
above-mentioned conditions. The cells were washed with PBS (2 ¥
1.0 mL), and then polymer 6 dissolved in Opti-MEM was added
◦
to the cells. After incubation at 37 C for 10 min, the cells were
washed with PBS (2 ¥ 1.0 mL), then WGA-QDs (2 mL in stock
solution, 1 mM) were added to the cells. The cells were incubated
at 37 ◦C for 1 h, washed with PBS (2 ¥ 1.0 mL), then Opti-MEM
(2.0 mL) and Hoechst 33342 (2.0 mL) were added. After washing
with PBS (2 ¥ 1.0 mL), the fluorescence was measured by CLSM.
maintained at 25.0
0.1 ◦C. All the solutions were carefully
protected from air by a stream of humidified argon. The degree of
protonation of the polymers was determined using the program
BEST. The total nitrogen content of the samples were determined
by elemental analysis and used to normalize the curves to the
degree of protonation.
Acknowledgements
This work was supported by an Industrial Technology Research
Grant Program in 2006 from the New Energy and Industrial
Technology Development Organization (NEDO) of Japan. The
flow cytometry analysis was carried out at the OPEN FACILITY,
Hokkaido University, Sousei Hall and HINTS.
Cell culture
HeLa cells were grown in a monolayer in Dulbecco’s modified
Eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), penicillin (500 units mL-1), and streptomycin
(500 mg mL-1). HT1080 cells were grown in DMEM-F12 medium
supplemented with 10% FBS, penicillin (500 units mL-1), and
streptomycin (500 mg mL-1) and TIG1-20 cells were grown in
DMEM-F12 supplemented with 10% FBS. The cultures were kept
at 37 ◦C in a humidified incubator under 5% CO2.
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