Enhancing cell recognition by scrutinizing cell surfaces with a nanoparticle array

Article abstract of DOI:10.1021/ja108527y

We report a dual-ligand nanoparticle array approach for discerning cells that have different surface receptor profiles surrounding a common primary receptor expressed at high or low levels. The achieved differentiation provides nanoparticles the ability for potential applications in treatment of patients at a personalized medicine level for drug delivery and radiation therapy with a much better safety profile.

Full text of DOI:10.1021/ja108527y

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Enhancing Cell Recognition by Scrutinizing Cell Surfaces with a
Nanoparticle Array
Hongyu Zhou,† Peifu Jiao,†,‡ Lei Yang,† Xi Li,† and Bing Yan*,†,‡
†Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States
‡School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
S Supporting Information
b
enhance cell recognition and, more importantly, differentiate
ABSTRACT: We report a dual-ligand nanoparticle array
approach for discerning cells that have different surface
receptor profiles surrounding a common primary receptor
expressed at high or low levels. The achieved differentiation
provides nanoparticles the ability for potential applications
in treatment of patients at a personalized medicine level for
drug delivery and radiation therapy with a much better
safety profile.
these similar cells having a common receptor such as FRs. To test
this hypothesis, a DLNA containing 30 members was synthe-
sized. To achieve the maximum surface structural diversity in this
DLNA, we selected highly diverse building blocks, including five
acylators and six amines. Every folic acid (FA) ligand in this array
was surrounded by secondary ligands, which contained diverse
molecular structures from combinatorial library synthesis
(Figure 1a; detailed information on the synthesis of the second
ligand library, thioctic-FA, and DLNA is included in the Support-
ing Information). Our previous works have shown that combi-
natorial chemistry modifications on surfaces of nanoparticles
altered their interactions with proteins and cells.20,21 Members of
the array were characterized by transmission electron micro-
scopy (TEM; Figure S1 in the Supporting Information), UV-
vis spectrometry (Figure S2), dynamic light scattering (DLS;
Figure S3), and ζ potential measurements (Table S1 in the
Supporting Information). The ratio of the secondary ligand to
FA was between 7 and 12 (Table S2), as determined by iodine
cleavage and a quantitative HPLC/MS/UV/chemiluminescent
nitrogen detection (CLND) method (Figure S4).22 After integ-
rity confirmation, we examined their cell recognition capabilities
by inductively coupled plasma mass spectroscopy (ICP-MS)
quantification of GNPs bound to or taken up by cells.
Alternating secondary ligands surrounding FA resulted in
different cell recognition patterns, as shown by cell binding and
uptake of GNPs (Figure 1b-d and Figures S5-S7). The cell
binding and uptake of dual-ligand GNPs was expressed by the
relative amounts of cellular gold in comparison with that from
binding of monoligand (FA) GNPs (GNP-FA; Figure 1b-d).
From the heat maps in Figure 1b-d, D55 and D56 exhibited cell
recognition enhancement by a factor of 3-4 for Hela cells, while
D16 and D21 exhibited the highest enhancement effects for
HepG2 cells. Diverse secondary ligands on dual-ligand GNPs
generated different cell recognition patterns, suggesting that the
microenvironmental receptor profiles surrounding FRs in these
cells are indeed different and that the DLNA approach is highly
effective in identifying selective ligands when the cell receptor
profile is unknown.
ell-cell recognition through multivalent interactions be-
C
tween multiple ligand-receptor pairs is an ideal model for
nanoparticle cell delivery systems. The current nanoparticle
systems achieve cell recognition by functionalization of the
nanoparticles with a targeting ligand such as antibodies,1,2
oligopeptides,3,4 nucleic acid aptamers,5-7 carbohydrates,8-10
or other molecules.11,12 However, a dilemma is that the single-
receptor targeting cannot differentiate cells that happen to have a
common receptor, such as cells from different patients with the
same disease. Another serious problem is that a receptor over-
expressed in diseased cells (e.g., cancer cells) is also expressed at a
lower level in normal cells, so a single-ligand targeting system
may have off-target effects on normal cells.13,14 Since cells express
multiple surface receptors, the dual-targeting approach has been
considered an improvement for cell recognition. Although
results from the proof-of-concept experiments with two known
receptor ligands have been evaluated,15-19 this approach is
severely hindered in general by the lack of receptor information
on the cell surface. Here we address these critical issues by the
development of highly selective nanoparticles decorated with
dual-targeting molecules through scanning of the cell surface
with a dual-ligand nanoparticle array (DLNA) that varies
the molecular diversity of the secondary ligands surrounding
the primary ligand on gold nanoparticles (GNPs). We demon-
strate below that this DLNA can discern the slightest difference
between cells that have different surface receptor profiles sur-
rounding a common primary receptor expressed at high or low
levels.
Comparison of the cell recognition of the dual-ligand GNPs
with that of each of the monoligand GNPs (M is used to indicate
GNPs with only the secondary ligand) revealed a 3-10-fold
increase of cell binding and uptake for all cells was observed
(Figure 2a,b). This enhancement was much more than an
Cervical cancer cells (Hela), epidermal cancer cells (KB), and
hepatocellular carcinoma cells (HepG2) all overexpress folate
receptors (FRs). However, their surface receptor profiles sur-
rounding FRs are likely to be different because of their different
origins. We hypothesized that ligands that interact with the
secondary receptors surrounding FRs for these cell lines would
Received: September 21, 2010
Published: December 23, 2010
r
2010 American Chemical Society
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dx.doi.org/10.1021/ja108527y J. Am. Chem. Soc. 2011, 133, 680–682
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Figure 2. (a, b) Cellular uptake of selected hits and monoligand GNPs
for (a) KB and (b) HepG2 cells. (c) Cellular uptake of M55 and D55 for
Hela cells in the presence of either ligand 55 or free FA. The GNP
concentration for all experiments was 50 μg/mL. The data represent
means ( standard deviations of results from three experiments. The
labels * and ** indicate P < 0.05 and P < 0.01, respectively, vs D55, as
determined by Student's t test.
that had high and low expressions of a common primary receptor
with different surface receptor profiles around the primary
receptor. This was accomplished by scanning the surfaces of
Hela and A549 cells with the array. A549 cells express very low
levels of FR and should have different surrounding receptors than
in Hela cells because of their different organ origin. As shown
above, scanning the surface of the Hela cells identified D55 as a
GNP that binds with high affinity. The binding of GNP-FA to
Hela cells was enhanced by 100% relative to its binding to A549
cells. With dual-ligand GNP D55, the cell binding to Hela cells
was enhanced by 400% relative to its binding to A549 cells
(Figure 3a). Since cellular uptake of GNPs may promote the
detrimental effects of X-ray radiation on cell viability,23-26 we
validated this enhancement by comparing the radiation-induced
cell death in these two cell lines. Even at the highest dose of D55
(150 μg/mL), X-ray irradiation (130 kV for a single dose of
10 Gy) did not induce a detectable amount of cell death in A549
cells (Figure 3b). The same dose of X-rays induced 60% cell
death in FR-positive Hela cells (Figure 3b). Our data showed that
X-ray radiation selectively killed cells with high levels of FRs
(Hela) but did not harm cells with low levels of FRs (A549).
In summary, to mimic multivalent cell-cell recognition, we
employed a dual-ligand combinatorial GNP array to scrutinize
the cell surface to identify the most selective nanoparticles.
Selected nanoparticles distinguish cells that have a common
receptor at high or low expression levels with different surround-
ing receptor profiles. The sensitive differentiation of cells with
high expression of a common primary receptor and different
secondary receptor profiles potentially provides an opportunity to
treat patients at a personalized medicine level. The enhancement of
Figure 1. (a) Surface molecular compositions of dual-ligand GNP array
members. (b-d) Heat maps showing the relative cellular uptake
amounts of dual-ligand GNPs for (b) Hela, (c) KB, and (d) HepG2
cells in comparison with GNP-FA. The GNP concentration for all
experiments was 50 μg/mL.
additive effect, suggesting a possible cooperative multivalent
effect. We compared the cell binding with the computed LogD
at pH 7.4 and molecular surface area, and no correlations were
found, excluding the possibility that the enhancement was due to
nonspecific interactions from the secondary ligands (Figure S8).
To further investigate whether the specific interactions between
the secondary ligands and cell surface receptors were responsible
for the enhancement, we preincubated cells with free FA, ligand
55, or both before adding D55. The competing free ligands
effectively blocked the cell binding for Hela cells (Figure 2c),
indicating that the enhanced cell recognition was likely due to the
cooperative binding of both ligands to their respective receptors.
Besides the sharp differentiation of cells with an overexpressed
common receptor, the DLNA was also used to differentiate cells
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dx.doi.org/10.1021/ja108527y |J. Am. Chem. Soc. 2011, 133, 680–682

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’ ASSOCIATED CONTENT
(21) Zhang, B.; Xing, Y. H.; Li, Z. W.; Zhou, H. Y.; Mu, Q. X.; Yan, B.
Nano Lett. 2009, 9, 2280–2284.
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Supporting Information. Experimental section; charac-
b
terization of dual-ligand GNPs by UV-vis spectroscopy, TEM,
DLS, ζ potential measurements, and HPLC/MS/UV/CLND;
and cellular uptake of dual-ligand GNPs in Hela, KB, and HepG2
cells. This material is available free of charge via the Internet at
http://pubs.acs.org.
(23) Liu, C. J.; Wang, C. H.; Chien, C. C.; Yang, T. Y.; Chen, S. T.;
Leng, W. H.; Lee, C. F.; Lee, K. H.; Hwu, Y.; Lee, Y. C.; Cheng, C. L.;
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’ AUTHOR INFORMATION
Corresponding Author
dr.bingyan@gmail.com
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’ ACKNOWLEDGMENT
We thank Sharon Frase for TEM images and Thomas
Mohaupt and Paul Thomas for assistance in X-ray experiments.
This work was supported by the National Basic Research
Program of China (973 Program, 2010CB933504), the National
Natural Science Foundation of China (21077068), the National
Cancer Institute (P30CA027165), the American Lebanese
Syrian Associated Charities (ALSAC), and St. Jude Children's
Research Hospital.
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