Cell compatible trimethoprim-decorated iron oxide nanoparticles bind dihydrofolate reductase for magnetica ly modulating focal adhesion of mammalian cells

Article abstract of DOI:10.1021/ja202767g

On the basis of the high affinity binding of trimethoprim (TMP) to Escherichia coli dihydrofolate reductase (eDHFR), TMP-decorated iron oxide nanoparticles bind to eDHFR with high affinity and specificity, which allows magnetic modulation of focal adhesio

Full text of DOI:10.1021/ja202767g

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Cell Compatible Trimethoprim-Decorated Iron Oxide Nanoparticles
Bind Dihydrofolate Reductase for Magnetically Modulating Focal
Adhesion of Mammalian Cells
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Marcus J. C. Long,†, Yue Pan,‡, Hsin-Chieh Lin,‡ Lizbeth Hedstrom,‡,§ and Bing Xu*,‡
†Graduate Program in Biochemistry and Biophysics, ‡Department of Chemistry, and §Department of Biology, Brandeis University,
415 South Street, Waltham, Massachusetts 02454, United States
S Supporting Information
b
possible choices of ligand/receptor pairs (e.g., biotin/avidin,13
GSH/GST,14 TMP/eDHFR,15 and amylose/maltose binding
ABSTRACT: On the basis of the high affinity binding of
trimethoprim (TMP) to Escherichia coli dihydrofolate reductase
protein (MBP)16), we chose trimethoprim (TMP) as the ligand
(eDHFR), TMP-decorated iron oxide nanoparticles bind to
eDHFR with high affinity and specificity, which allows magnetic
modulation of focal adhesion of mammalian cells adhered to a
surface. Besides being the first example of nanoparticles that
selectively bind to eDHFR, the biocompatibility of the conjugate
of TMPÀiron oxide nanoparticles renders a convenient and
versatile platform for investigating the cellular responses to
specific, mechanical perturbation of proteins via a magnetic force.
and E. coli dihydrofolate reductase (eDHFR) as the receptor
because of the high affinity (Kd = 9 nM) of TMP exhibited for the
bacterial DHFR.15 Although there are agaroseÀmethotrexate
microbeads, they bind to both human DHFR and bacterial
DHFR indiscriminately, and methotrexate on iron oxide mag-
netic nanoparticles retains its cytotoxicity.17 With respect to the
compatibility/functionality with mammalian cells, trimethoprim
is more advantageous because its toxicity in mammalian cells is
lower than that of methotrexate. More importantly, the affinity of
TMP for mammalian DHFR homologues is about 50000 times
lower than for eDHFR, thus allowing selective manipulation of
an ectopically expressed eDHFR fusion protein in a mammalian
cell. Thus, TMP/eDHFR is considered to be a bio-orthogonal
system in eukaryotic cells that has been used successfully on
many occasions.15,18 In addition, the binding between TMP and
eDHFR is monovalent, which disfavors the cross-link that usually
occurs in the biotin/avidin-based systems. The orthogonality,
monovalency, and small size of TMP offer a tremendous advan-
tage for TMP/eDHFR pair over many other affinity systems,
thus making TMP an ideal candidate for decorating iron oxide
nanoparticles for biomedical applications.
On the basis of the above rationale, we used the recently developed
dopamine anchors7,19 to attach TMP to iron oxide nanoparticles
(∼6 nm in diameters) for binding eDHFR fusion proteins and
manipulating cells. Our results show that TMP-functionalized nano-
particles exhibit ultrahigh affinity for eDHFR fusion proteins. This
interaction is highly specific and has a long half-life, that is, behaves as
an (almost) irreversible interaction under normal (nondenaturing)
conditions. Most importantly, these TMP-decorated nanoparticles
are cell compatible (94% cell viability after 48 h exposure to
TMP decorated nanoparticles) and have the ability to alter the focal
adhesion of the cells expressing the eDHFR fusion proteins in vitro.
Since focal adhesion, as a fundamental cellular process, has important
implications for development, differentiation, disease, and regenera-
tion,20 this result is significant because it offers a simple and viable
process for investigating cellular responses to mechanical perturbation
of specific proteins via magnetic (i.e., noncontact) modulation.
Scheme 1 shows a simple procedure to anchor TMP on an
iron oxide magnetic nanoparticle. We first synthesized 1 from
trimethoprim via the selective demethylation of the para ether
mong a large variety of nanomaterials, magnetic nanoparticles1À3
A
have received considerable attention in the past decade because
they offer precisely controlled size, ability to respond to noncontact
manipulation, and enhancement of contrast in magnetic resonance
imaging (MRI). As a result, magnetic nanoparticles promise many
applications in biology and medicine, including medical imaging,4,5
protein purification,6À8 bacteria capture,9 and drug delivery.10 Parti-
cularly, because of their inherent biocompatibility, iron oxide mag-
netic nanoparticles have become the leading candidate for developing
biofunctional molecular imaging agents for targeted imaging and
therapy.4,11 Despite the considerable advances in the preparation of
iron oxide magnetic nanoparticles,1 their biofunctionalization still
suffers several shortcomings. For example, it is quite common to
attach poly(ethylene glycol) (PEG) on the surface of iron oxides for
reducing unwanted accumulation of the nanoparticles in liver or
spleen in vivo, but this attachment also decreases the binding
efficiency of the nanoparticles to their targets. In principle, the link
of antibodies onto the nanoparticles can increase specificity, but it is
nontrivial to control the orientation and the number of antibodies on
the nanoparticles. On the other hand, the decoration of the coordina-
tion complex of nickel-nitrilotriacetic acid (Ni-NTA) on magnetic
nanoparticles, via a dopamine or other linkers,6,7 has achieved high
specificity and capacity. The potential cytotoxicity associated with
nickel, however, prevents the direct use of Ni-NTA on live cells for
long duration.12 These limitations require the development of a new
approach to present proteins on magnetic nanoparticles without
compromising the binding affinity, biological compatibility, and
molecular orientation of the proteins.
To satisfy the above requirements, one promising candidate is
the small-molecule ligand/receptor pairs that have already gained
popularity in protein pull-down assays, which imply appropriate
orientation of the proteins on the surface of the particles after the
binding between the ligands and the receptors. Among several
Received: March 27, 2011
Published: June 10, 2011
r
2011 American Chemical Society
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Scheme 1. Synthetic Route of the TMP-Decorated Iron
Oxide Nanoparticles
Figure 2. SDS/PAGE analysis (by silver staining24) of (A) binding of 4
to GFP-TEV-HA-eDHFR in a E. coli cell lysate: lane 1, cell lysate; lane 2,
elution by 2Â sds loading buffer at 60 °C for 4 h; lane 3, molecular
weight marker and (B) binding of 4 to eDHFR-HA-GFP in a mamma-
lian cell lysate: lane 4, cell lysate; lane 5, elution by 2Â sds loading buffer
at 60 °C for 4 h; lane 6, molecular weight marker.
20À30 μm) suggests that it is possible to use magnetic nano-
particles to manipulate cellular components (e.g., ribosomes),
cellular organelles (e.g., nucleus), and cells via a magnetic force.
However, to realize the magnetic control of such a wide range of
targets, 4 has to meet two basic prerequisites: (i) selectively
binding eDHFR fusion proteins and (ii) being cell compatible.
We first confirmed that 4 can specifically bind eDHFR fusion
proteins. After incubating 4 with the lysate of E. coli overexpres-
sing GFP-TEV-HA-eDHFR for 2 h in PBS (TEV is a protein
sequence which recognized by tobacco etch virus protease; HA is
a protein epitope derived from hemagglutinin), we used a magnet
to attract nanoparticles to the wall of the vial and washed them
twice with PBS, and then eluted the protein using loading buffer
(LB) containing 4% sodium dodecylsulfate (SDS) at 60 °C for
4 h. As shown in Figure 2A, elution by LB at 60 °C results in one
band (lane 2), which is the fusion protein GFP-TEV-HA-
eDHFR (confirmed by anti-HA Western blot21). Meanwhile,
no protein was eluted by LB at room temperature. These results
confirm that 4 selectively and tightly binds to GFP-TEV-HA-
eDHFR from the E. coli lysate. We also used the same conditions
to bind the fusion protein, eDHFR-HA-GFP, expressed in a
mammalian cell (HeLa). As shown in Figure 2 B, elution by LB at
60 °C gives one band (lane 5), which is eDHFR-HA-GFP. Since
the HeLa cell expresses the eDHFR-HA-GFP at relatively low
levels against a noisy background,21 this result further confirms
the high specificity of 4 to eDHFR. After being separated from
the lysates, the conjugates, GFP-TEV-HA-eDHFRÀ4, exhibit
green fluorescence,21 confirming that the GFP proteins preserve
their innate properties after being linked to the nanoparticles by
fusing with eDHFR.
After confirming the binding efficiency of 4 to eDHFR, we
assessed the biocompatibility of the nanoparticles toward mam-
malian cells. After 2 days and 5 days incubation with different
amount of 4, COS-1 and HeLa cells received a 50% trypan blue
solution as a negative stain for live cells.25 As shown in A and B of
Figure 3, more than 90% of the cells are viable even after 5 days
incubation with 200 μg/mL of 4, suggesting that 4 is compatible
with the cells. Furthermore, after incubating the cells with 4 for
7 days, we transfected the cells with a plasmid expressing a
monomeric red fluorescence proteinÀglutathione-S-transferase
fusion protein (mRFPÀGST) and found that these cells success-
fully express mRFPÀGST.21 These results prove that cells are
viable after prolonged exposure to the TMP-decorated iron oxide
Figure 1. Possible binding of 4 with eDHFR protein on the nanopar-
ticles; and relative size of 4, a ribosome, and a cell.22
group and the subsequent attachment of a methylene carboxylate
group. Then we coupled the dopamine (2) with 1 via HBTU and
DIEA in DMF to generate 3 (yield =31%). Finally, iron oxide
nanoparticles react with 3 in phosphate buffer at the pH of 6.0 to
afford the TMP-functionalized iron oxide nanoparticles (4).
Transmission electron micrograph reveals that the nanoparticles
of 4 have a diameter of about 6 nm, which is similar to that of as-
prepared iron oxide nanoparticles.21 Interestingly the attachment
of TMP on the iron oxide nanoparticles barely induces the
aggregation of these particles in water, likely due to the hydro-
philic character of the diaminopyrimidine of TMP. Besides, the
IR spectra21 of 4 exhibit a broad peak around 3340 cmÀ1 and a
strong sharp peak centered at 1670 cmÀ1, which originate from
NÀH (from NH2) and CdO (from amide) stretch vibrations,
respectively, thus indicating the presence of 3 on 4. In addition,
weight analysis gives the estimation of about 37 molecules of 3 on
each iron oxide nanoparticle.21
As shown in Figure 1, a simple molecular docking model based
on the lattice of magnetite3 and the crystal structure of the
complex of TMP and eDHFR23 suggests that it is possible for 4
to bind the eDHFR protein without a spacer between dopamine
and TMP. Because of the robust binding between dopamine and
iron oxide,7,19 the conjugate of 4 and eDHFR should be
kinetically stable, which likely results in a relatively constant
orientation of eDHFR on the surface of the nanoparticles for
presenting other proteins via fusion at the (N or C) terminus of
eDHFR. Most importantly, the comparable sizes between
eDHFR and the nanoparticle indicate that the nanoparticles
should minimize unwanted nonspecific interactions usually
associated with microparticles because the target proteins can
cover the surface of the nanoparticles effectively and quickly.
In addition, the comparison of the diameters of 4 (6 nm), a
ribosome (20À30 nm), and a cell (e.g., COS-1 cell is about
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from the surface. Thus, 4 permits magnetic modulation of focal
adhesion of live cells. This effect is absent in a control group of cells
(not incubated with 4).21 Given that eDHFR and GFP are not
expected to associate strongly with mammalian proteins, the strong
attraction of COS-1 incubated with 4 to a magnet can account for
this effect.
In conclusion, we used biocompatible TMP-decorated iron
oxide magnetic nanoparticles to bind eDHFR fusion proteins
from cell lysate and further magnetically modulate focal adhesion
of live cells. The use of nanoparticles for binding a specific
protein from the milieu of a cell lysate opens many opportunities,
ranging from ectopic protein delivery to magnetic manipulation
of cellular functions because, unlike microbeads, nanoparticles
undergo endocytosis easily. Although the mechanism of the
magnetic modulation of focal adhesion has yet to be established,
the further development of this approach ultimately may allow
the control of the spatiotemporal behavior of proteins or sub-
cellular organelles, which should provide a new way to help
validate functional impact of the locations of proteins and
contribute to establishing and regulating molecular pathways
for a wide range of cellular functions.
Figure 3. Viability of (A) COS-1 and (B) HeLa cells incubated with
different amount of 4 (15, 25, 90, and 200 μg/mL) after 2 days and
5 days (tested by a 50% trypan blue solution).
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental sections, NMR,
b
LCÀMS, sequence of GFP-TEV-HA-eDHFR and eDHFR-HA-
GFP protein, infrared spectra, TEM, fluorescent image, SDS-
PAGE analysis, anti-HA Western blot, and the video of cell
migration. This material is available free of charge via the Internet
at http://pubs.acs.org.
Figure 4. Magnetically modulated focal adhesion of COS-1 cell for 4 h.
(A) t = 0 h (elongate forming a diagonal (45°) axis running upward from
right to left), (B) t = 1.5 h (shift down slightly and the axis move to 20°),
(C) t = 3 h (twist and run vertically, overall a 45° rotation), (D) t = 4 h
(detached, no longer visible). The relative position of the magnet versus
the cell is shown in top view (scale bar =10 μm).
’ AUTHOR INFORMATION
Corresponding Author
bxu@brandeis.edu
nanoparticles and the uptake of 4 causes very few adverse effects
to the protein expression in the cells.
Author Contributions
These authors contributed equally.
Though pioneering research has been carried out by employ-
ing magnetic microbeads to study focal adhesion,26 the corre-
sponding investigations using nanomaterials have not been
undertaken. Thus, we decided to use 4 to perturb the focal
adhesion of cells. After establishing the cell compatibility of 4, we
showed that cells incubated with 4 were attracted to a magnet
when trypsinized.21 Subsequently, we incubated COS-1 cells,
transiently transfected with eDHFR-HA-GFP, with 25 μg of 4 in
1 mL complete medium for overnight.21 As shown in Figure 3A, the
concentration of 4 used (25 μg) has been shown to have little effect
on cell viability even after 5 days incubation. After washing the cells
three times with Dulbecco’s PBS to remove unbound 4, we placed a
magnet (with surface magnetic field about 4000 G) and observed
the behavior (e.g., focal adhesion) of the cells over 4 h. As shown in
Figure 4, the cell adheres to the plate at the initial moment, showing
a characteristic asymmetric shape (nonadhered cells are typically
round), that is the cell elongates along a diagonal axis running
upward from left to right. After being under magnetic attraction for
1.5 h, the cell shifts down slightly so the axis of the cell tilts toward
the vertical direction (Figure 4B). After 3 h attraction, the cell twists
in such a way that its axis runs almost vertically (Figure 4C). After
4 h, the cell detaches from the surface and moves out of the view
field (Figure 4D). Under the magnetic attraction for 4 h, the iron
oxide magnetic nanoparticles in the cells clearly exert a mechanical
force on the cell to cause cell-shape distortion and cell detachment
’ ACKNOWLEDGMENT
We acknowledge the financial support from a start-up grant
from Brandeis University. Y.P. thanks the Brandeis EM facility
for assistance. We also thank Rafael A. Cabanas for static light
scattering measurement.
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