Facile surface functionalization of hydrophobic magnetic nanoparticles

Article abstract of DOI:10.1021/ja5060324

Nonpolar phase synthesized hydrophobic nanocrystals show attractive properties and have demonstrated prominent potential in biomedical applications. However, the preparation of biocompatible nanocrystals is made difficult by the presence of hydrophobic surfactant stabilizer on their surfaces. To address this limitation, we have developed a facile, high efficiency, single-phase and low-cost method to convert hydrophobic magnetic nanoparticles (MNPs) to an aqueous phase using tetrahydrofuran, NaOH and 3,4-dihydroxyhydrocinnamic acid without any complicated organic synthesis. The as-transferred hydrophilic MNPs are water-soluble over a wide pH range (pH = 3-12), and the solubility is pH-controllable. Furthermore, the as-transferred MNPs with carboxylate can be readily adapted with further surface functionalization, varying from small molecule dyes to oligonucleotides and enzymes. Finally, the strategy developed here can easily be extended to other types of hydrophobic nanoparticles to facilitate biomedical applications of nanomaterials.

Full text of DOI:10.1021/ja5060324

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Communication
Facile Surface Functionalization of Hydrophobic Magnetic Nanoparticles
Yuan Liu, Tao Chen, Cuichen Wu, Liping Qiu, Rong Hu, Juan Li, Sena Cansiz,
Liqin Zhang, Cheng Cui, Guizhi Zhu, Mingxu You, Tao Zhang, and Weihong Tan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja5060324 • Publication Date (Web): 20 Aug 2014
Downloaded from http://pubs.acs.org on August 25, 2014
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Facile Surface Functionalization of Hydrophobic Magnetic
Nanoparticles
Yuan Liu, Tao Chen, Cuichen Wu, Liping Qiu, Rong Hu, Juan Li, Sena Cansiz, Liqin Zhang, Cheng
Cui, Guizhi Zhu, Mingxu You, Tao Zhang, Weihong Tan*
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$
$
Center for Research at Bio/Nano Interface, Department of Chemistry and Department of Physiology
and Functional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute,
University of Florida, Gainesville, Florida 32611-7200, United States
ABSTRACT: Nonpolar phase synthesized hydrophobic nanocrystals show attractive properties and have demonstrated prominent
potential in biomedical applications. However, the preparation of biocompatible nanocrystals is made difficult by the presence of
hydrophobic surfactant stabilizer on their surfaces. To address this limitation, we have developed a facile, high efficiency, single-
phase and low-cost method to convert hydrophobic magnetic nanoparticles (MNPs) to an aqueous phase using tetrahydrofuran,
NaOH and 3, 4-dihydroxyhydrocinnamic acid without any complicated organic synthesis. The as-transferred hydrophilic MNPs are
water soluble over a wide pH range (pH=3 to 12), and the solubility is pH-controllable. Furthermore, the as-transferred MNPs with
carboxylate can be readily adapted with further surface functionalization, varying from small molecule dyes to oligonucleotides and
enzymes. Finally, the strategy developed here can easily be extended to other types of hydrophobic nanoparticles to facilitate bio-
medical applications of nanomaterials.
Hydrophobic nanocrystals synthesized in nonpolar sol-
vents by high-temperature thermolysis show attractive proper-
ties, such as tunable size with narrow size distribution, and
labor-intensive, thus making the ligand exchange method more
complicated. Amphiphilic ligand encapsulation is simple, but
it may not result in stable MNPs by the noncovalent hydro-
phobic interactions, and additional organic syntheses are nec-
essary as well.
1,2
low crystalline defect. Among these materials, magnetic
nanoparticles (MNPs), because of their unique nanoscale
physical and chemical properties, have demonstrated promi-
nent potential in biomedical applications, including magnetic
3
-5
6,7
resonance imaging (MRI), targeted drug delivery, and
8
-11
hyperthermia for cancer treatment.
Despite their success in biomedical science, the prepara-
tion of biocompatible MNPs is made difficult by the presence
of a hydrophobic surfactant stabilizer on their surfaces. To
solve this problem, many strategies have been developed to
transfer the hydrophobic MNPs to an aqueous phase. One
popular method is ligand exchange, in which small molecules
are used to replace the hydrophobic ligand. The other is am-
phiphilic ligand encapsulation, which involves formation of a
hydrophilic shell on the surface of MNPs. Unfortunately, the
ligand exchange method is typically performed in two phases:
Scheme 1. Ligand exchange using tetrahydrofuran, 3, 4-
dihydroxyhydrocinnamic acid (DHAC) and NaOH.
To address these obstacles, we have developed a facile,
high efficiency, single-phase and low-cost method to transfer
hydrophobic MNPs to an aqueous phase over a wide pH range
(pH=3 to 12) using 3, 4-dihydroxyhydrocinnamic acid
(DHCA) in tetrahydrofuran (THF). As shown in Scheme 1, the
surfactant stabilizer oleic acid was replaced by DHCA, which
forms a robust anchor on the surface of magnetic nanoparticles
via a five-membered metallocyclic chelate. The MNPs were
then neutralized with NaOH to precipitate the sodium salt,
which is not soluble in THF. The ionic form was then dis-
persed in aqueous solution at moderate concentrations. The
resulting water-soluble MNPs were very stable over a wide pH
range from 3 to 12 (Figure S4 and S5 in Support Information
(SI)). In addition, MNPs coated with DHCA can be robustly
functionalized via the carboxyl group to form a peptide link-
MNPs in the hydrophobic phase and small molecules in the
5,12,13
hydrophilic phase.
For example, dopamine hydrochloride
14-16
has been used for ligand exchange on MNPs.
However,
because of the lack of solubility in nonpolar solvents, a two-
phase system is needed. Furthermore, MNPs tend to aggregate
when mixed with polar solvents. Thus, the exchange efficien-
cy is typically low because of inefficient interaction between
the MNPs and small molecules, and further aggregation may
occur from the low exchange ratio. In addition, dopamine-
transferred MNPs have poor biocompatibility as a result of
poor solubility in biological buffers. Although scientists have
tried various modifications, such as the addition of PEG, to the
small molecules to improve the stability of MNPs, extensive
organic syntheses are needed, which are time-consuming and
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age with other amine-containing molecules, varying from
used as solvent and no obvious aggregation was observed over
a period of 3 months.
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small molecule dyes to oligonucleotides and even enzymes.
Typically, DHCA without modification was dissolved in THF
in a three-necked flask. Hydrophobic MNPs (27.6±1.5 nm)
were added dropwise at 50 °C and kept for 3 hours at this
temperature. Upon cooling the reaction mixture, NaOH (0.5
M) was added to precipitate the MNPs, which were collected
by centrifugation and redispersed in water. The as-transferred
MNPs were spherical and fairly monodisperse without aggre-
gation, as shown by transmission electron microscopy (TEM)
images (Figure 1) and dynamic light scattering (DLS) (Figure
S7, SI).
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Figure 2. (A) pH-dependent solubility of MNPs and (B)
schematic illustration of pH controlled reversible aggregation
and dissociation of MNPs.
The DHCA anions not only offer MNPs excellent water
solubility but also provide a platform for further surface func-
tionalization via the carboxyl group to form a peptide linkage
with other amine-containing molecules. Therefore, we system-
atically investigated surface functionalization with fluoresce-
inamine (FLAM), a DNA aptamer, and an enzyme. FLAM
was chosen as a model small-molecule probe to test MNP
surface functionalization because FLAM has an amine group
which can form a peptide bond with the carboxyl group by 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC)/N-hydroxysuccinimide (NHS) coupling to give emis-
sion at 519 nm. In order to rule out the possibility of physical
adsorption, a control experiment was conducted with and
without EDC/NHS coupling. Fluorescence measurements
(Figure S8, SI) showed that MNP and MNP/FLAM without
EDC/NHS coupling do not fluoresce, while MNP/FLAM with
the EDC/NHS crosslinking gives a significant fluorescent
signal at 519 nm, indicating that FLAM was covalently linked
onto the surface of MNPs, and that the carboxylated MNP
surface could serve as a platform for robust biomolecule func-
tionalization.
Figure 1. TEM images of magnetic nanoparticles before lig-
and exchange in chloroform (A) and after ligand exchange in
water (B). Scale bar is 200 nm. Solvent dispersity of MNPs
(C) before and (D) after ligand exchange.
After ligand exchange, water with pH values ranging
from 1 to 12 was used to test the solubility and stability of the
as-transferred MNPs. MNPs aggregated when the pH was 2 or
lower (Figure S4, SI). When NaOH (0.5 M) was introduced to
the aggregated MNPs (pH=2) to raise the pH to 7, the aggre-
gated MNPs dissociated and redissolved, and the soluble
MNPs aggregated when HCl (0.1 M) was added to decrease
the pH to 2 or lower (Figure 2). Thus, the aggregation and
dispersion of MNPs is reversible by controlling the pH.
Having determined that the as-transferred MNPs are sta-
ble in biological systems, we next established their utility in
biomedical applications. Amine-modified Sgc8 aptamer
17
(ASAT) (see Table S1 for all DNA sequences ) labeled with
carboxytetramethylrhodamine (TAMRA) was used to modify
MNPs based on EDC/NHS coupling and to test their binding
ability to target cancer cells. In this study, we designed an
amine-modified random oligonucleotide library labeled with
TAMRA (ALT) as control to ASAT (Table S1, SI). Selected
from a large library by SELEX (Systematic Evolution of Lig-
ands by Exponential Enrichment), aptamers are single-
stranded oligonucleotides, which specifically bind to their
targets (CEM cells for Sgc8) by folding into distinct secondary
Isoelectric point precipitation (IPP) was introduced to ex-
plain the reversibility of MNP aggregation. IPP is the pH at
which the net primary charges of a protein become zero, lead-
ing to aggregation from reduced electrostatic repulsions. The
IPP value for MNPs was experimentally determined to be 2
according to the solubility test above. To prove this hypothe-
sis, the zeta-potentials of as-transferred MNPs at different pH
values were measured (Figure S6, SI). Consistent with the
solubility test, the results showed that the zeta-potential of an
MNP is -1.4 mV at pH 2. Addition of NaOH makes the MNP
surfaces more negative, thus increasing the electrostatic repul-
sion between the MNPs and allowing them to be dispersed in
water (Figure 2 (B)). Both TEM images and DLS results in
water with different pH values indicate that the MNPs were
monodisperse and stable for a period of several months. In
addition, to test the stability of as-transferred MNPs, either
phosphate buffered saline (PBS) or cell culture medium was
18
or tertiary structures. Ramos cells, which are not recognized
by Sgc8, were chosen as control cells. According to the flow
cytometry histogram (Figure 3), a large shift was observed for
CEM cells, while only a negligible shift was observed for Ra-
mos cells when treated with MNP-ASAT. For MNP-ALT, no
obvious shift was observed for either CEM or Ramos cells.
For target CEM cells, a slightly larger shift was observed for
MNP-ASAT compared to free ASAT. This can be attributed to
the multivalent effect of multiple aptamers on the surface of
MNP-ASAT, thus resulting in enhanced binding affinity to the
target cancer cells. Thus, the ASAT-modified MNPs have
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ligand exchange methods for nanocrystals, such as quantum
excellent binding ability on target cancer cells and can be used
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as specific fluorescence imaging agents.
dots and nanorods, and that the as-transferred nanoparticles
will find widespread application in nanobiotechnology.
ASSOCIATED CONTENT
Supporting Information.
Detailed synthesis and characterization of hydrophobic magnetic
nanoparticles, phase transfer, surface functionalization and de-
tailed experimental procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
tan@chem.ufl.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Figure 3. Flow cytometry histograms of CEM (target) and
Ramos (control) cells incubated with buffer only, ASAT,
MNP-ASAT, MNP-ALT, and ALT. The concentrations for all
probes are 1 nM.
The authors are grateful to Dr. Kathryn Williams for her critical
comments during the preparation of this manuscript. This work is
supported by grants awarded by the National Institutes of Health
(GM079359 and CA133086). It is also supported by grants
awarded by the National Key Scientific Program of China
Protein enzymes, while being widely used because of
their high biocatalytic activity, are limited by their low stabil-
ity and low recycling capability. Nanobiocatalysis, whereby an
enzyme is immobilized on a nanoparticle surface while retain-
ing its biocatalytic activity, is of significant importance for
(2011CB911000 and 2013CB933701), the Foundation for Innova-
tive Research Groups of NSFC (Grant 21221003), and China
National Instrumentation Program 2011YQ03012412.
REFERENCES
19,20
industrial reuse.
Therefore, we conducted a facile and ro-
(
1) Park, J.; An, K.; Hwang, Y.; Park, J.; Noh, H.; Kim, J.; Park, J.;
bust covalent enzyme immobilization on the MNP surface
using alkaline phosphatase (AP) as a model enzyme (Scheme
Hwang, N.; Hyeon, T. Nat. Mater. 2004, 3, 891-895.
(2) Lynch, J.; Zhuang, J.; Wang, T.; LaMontagne, D.; Wu, H.; Cao,
Y. C. J. Am. Chem. Soc. 2011, 133, 12664-12674.
(3) Perez, J. M.; Josephson, L.; O’Loughlin, T.; Hogemann, D.;
Weissleder, T. Nat. Biotechnol. 2002, 20, 816-820.
(4) Huh, Y.; Jun, Y.; Song, H.; Kim, S.; Choi, J.; Lee, J.; Yoon, S.;
Kim, K.; Shin, J.; Suh, J.; Cheon, J. J. Am. Chem. Soc. 2005, 127,
2
). The hydrolysis of p-nitrophenyl phosphate can be cata-
lyzed efficiently by AP when pH=9.8. The assay results (Fig-
ure S2, SI) indicated that MNP-APs possess excellent catalytic
activity. A 10-round catalytic recycle of MNP-AP demonstrat-
ed that MNP-AP retains its catalytic activity after many uses,
indicating that the as-transferred MNP is an excellent nano-
supporter for enzyme immobilization (Figure S3, SI).
1
2387-12391.
(
5) Chen, T.; Ocsoy, I.; Yuan, Q.; Wang, R.; You, M.; Zhao, Z.;
Song, E.; Zhang, X.; Tan, W. J. Am. Chem. Soc. 2012, 134,
13164-12167.
(6) Cheng, K.; Peng, S.; Xu, C.; Sun, S. J. Am. Chem. Soc. 2009,
31, 10637-10644.
1
(
7) Chen, T.; Shukoor, M. I. Wang, T.; Zhao, Z.; Yuan, Q.; Bam-
rungsap, S.; Xiong, X.; Tan, W. ACS Nano 2011, 5, 7866-7873.
8) Fortin, J.; Wilhelm, C.; Servais, J.; Menager, C.; Bacri, J.;
Gazeau, F. J. Am. Chem. Soc. 2007, 129, 2628-2635.
(
(9) Guardia, P.; Corato, R. D.; Lartigue, L.; Wilhelm, C.; Espinosa,
A.; Garcia-Hernandez, M.; Gazeau, F.; Manna, L.; Pellegrino, T.
ACS Nano 2012, 6, 3080-3091.
(10) Lee, J.; Jang, J. Choi, J.; Moon, S. H.; Noh, S.; Kim, J.; Kim, J.;
Kim, I.; Park, K. I.; Cheon, J. Nat. Nanotechnol. 2011, 6, 418-
422.
(11) Martinez-Boubeta, C.; Simeonidis, K.; Makridis, A.; Angelak-
eris, M.; Lglesias, O.; Guardia, P.; Cabot, A.; Yedra, L.; Estrade,
S.; Peiro, F.; Saghi, Z.; Midgley, P. A.; Conde-Leboran, I.;
Serantes, D.; Baldomir, D. Sci. Rep. 2013, 3, 1652-1659.
Scheme 2. Covalent MNP surface functionalization with
alkaline phosphatase.
In summary, we have developed a facile, high efficiency,
single-phase and low cost method for aqueous phase transfer
of hydrophobic MNPs using DHCA and THF. Without any
complicated organic synthesis, MNPs neutralized with NaOH
show excellent water solubility and stability in biological envi-
ronments, demonstrating that the approach is more cost-
effective and labor-efficient than the traditional two-phase
ligand exchange method. Moreover, we report the first hydro-
philic nanoparticles with wide pH-range solubility and pH-
controllable aggregation. The as-transferred MNPs with car-
boxylate can be readily adapted by further surface functionali-
zation which is simple and robust. Based on these superior
features, we believe that this method can be applied to other
(
12) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose,
S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss,
S. Science 2005, 307, 538-544.
(13) Tong, S.; Hou, S.; Ren, B.; Zheng, Z.; Bao, G. Nano Lett. 2011,
11, 3720-3726.
(14) Xu, C.; Xu, K.; Gu, H.; Zheng, R.; Liu, H.; Zhang, X.; Guo, Z.;
Xu, B. J. Am. Chem. Soc. 2004, 126, 9938-9939.
(
15) Shultz, M. D.; Reveles, J. U.; Khanna, S. N.; Carpenter, E. E. J.
Am. Chem. Soc. 2007, 129, 2482-2487.
(
16) Mazur, M.; Barras, A.; Kuncser, V.; Galatanu, A.; Zaitzev, V.;
Turcheniuk, K. V.; Woisel, P.; Lyskawa, J.; Laure, W.; Siriwar-
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Journal of the American Chemical Society
Page 4 of 5
dena, A.; Boukherroub, R.; Szunerits, S. Nanoscale, 2013, 5,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
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4
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4
5
5
5
5
5
5
5
5
5
5
6
2692-2702.
(
(
17) Shangguan, D.; Li, Y.; Tang, Z.; Cao, Z. C.; Chen, H. W.; Mal-
likaratchy, P.; Sefah, K.; Yang, C. J.; Tan, W. Proc. Natl. Acad.
Sci. U.S.A. 2006, 103, 11838-11843.
18) Sefah, K.; Shangguan, D.; Xiong, X.; O’Donoghue, M. B.; Tan,
W. Nat. Protoc. 2010, 5, 1169-1185.
(19) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat.
Biotechnol. 2004, 22, 211-213.
20) Sapsford, K. E.; Algar, W. R.; Berti, L.; Gemmill, K. B.; Casey,
(
B. J.; Oh, E.; Stewart, M. H.; Medintz, I. L. Chem. Rev. 2013,
1
13, 1904-2074.
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