Functionalization of iron oxide nanoparticles with a versatile epoxy amine linker

Article abstract of DOI:10.1039/c0jm00808g

A synthetically diverse linker molecule consisting of both a terminal epoxide and a terminal amine has been synthesized and shown to have the desired reactivity. Proof of principle experimentation showed that the prepared linker molecule possessed the ability to be reactive towards dextran coated iron nanoparticles, essentially converting the surface alcohols to amines with an efficiency on average of 50 linkers per nanoparticle. Once the surface of the nanoparticles had been functionalized, the iron nanoparticles were subsequently functionalized with both folic acid and fluorescein isothiocyanate, with an average efficiency of 20 and 3 molecules per nanoparticle, respectively. The labeled nanoparticles were then incubated with both folate receptor positive and negative cell lines, which showed a preferential accumulation of the particles in the receptor positive cell line. In addition to the fluorescence based assays, accumulation of the nanoparticles was demonstrated using T2-weighted MRI imaging, which showed that the iron core of the nanoparticle was present within the desired cell line. Overall, this linker has shown the ability to functionalize the surface of nanoparticles and can theoretically be used to label a wide variety of other targeting agents or imaging agents for in vivo therapies or diagnostics. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0jm00808g

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www.rsc.org/materials | Journal of Materials Chemistry
Functionalization of iron oxide nanoparticles with a versatile epoxy amine
linker
ab
Michael Nickels,a Jingping Xie,a Jared Cobb,ab John C. Goreab and Wellington Pham
*
Received 23rd March 2010, Accepted 26th April 2010
First published as an Advance Article on the web 6th May 2010
DOI: 10.1039/c0jm00808g
conjugation of native peptides, proteins, or antibodies. A number of
techniques have been developed for the functionalization of iron
oxide nanoparticles, including the in situ conversion of dextran
hydroxyl groups into amines, which is typically used for the encap-
sulation of iron core.10 In the recent work, Young et al. coated
iron nanoparticles with amine groups using a tripartite component
consisting of dopamine, polyethylene glycol (PEG), and
trichloro-s-triazine.11 Another approach to fabricating the surface of
iron nanoparticles with amine groups uses glycol chitosan.12
Additional examples include the coating of the iron core with
a,u-dicarboxyl-terminated PEG or poly(acrylic acid) to afford the
free surface carbonyl groups necessary for further covalent bonding
with the targeted molecules.13,14 Despite all of these achievements, the
development of functionalized nanoparticles for targeting imaging
still remains a significant problem. Preparation of such probes
requires synthetic steps, which make it difficult to analyze the final
products.
A synthetically diverse linker molecule consisting of both a terminal
epoxide and a terminal amine has been synthesized and shown to
have the desired reactivity. Proof of principle experimentation
showed that the prepared linker molecule possessed the ability to be
reactive towards dextran coated iron nanoparticles, essentially
converting the surface alcohols to amines with an efficiency on
average of 50 linkers per nanoparticle. Once the surface of the
nanoparticles had been functionalized, the iron nanoparticles were
subsequently functionalized with both folic acid and fluorescein
isothiocyanate, with an average efficiency of 20 and 3 molecules per
nanoparticle, respectively. The labeled nanoparticles were then
incubated with both folate receptor positive and negative cell lines,
which showed a preferential accumulation of the particles in the
receptor positive cell line. In addition to the fluorescence based
assays, accumulation of the nanoparticles was demonstrated using
T2-weighted MRI imaging, which showed that the iron core of the
nanoparticle was present within the desired cell line. Overall, this
linker has shown the ability to functionalize the surface of nano-
particles and can theoretically be used to label a wide variety of
other targeting agents or imaging agents for in vivo therapies or
diagnostics.
This paper reports the synthesis and properties of a versatile linker
for the surface modification of nanoparticles. The underlying prin-
ciple of this work is to generate a ready-to-use and water-soluble
linker equipped with a highly reactive functional group such as an
epoxide. The approach uses dextran-coated nanoparticles developed
in our laboratory as a template for such modifications.15 Since
hydroxyl groups on dextran are weak nucleophiles, the use of an
epoxide guarantees a neat reaction without the use of a catalyst to
enhance the simple reaction work-up. To our knowledge, this is the
first report to describe the synthesis of an amine epoxide linker to
generate aminated iron oxide nanoparticles.
The integration of nanotechnology with molecular imaging in the
past decade has made a remarkable impact on biomedical research.
In particular, the development of the iron oxide nanoparticles
exemplifies this noteworthy advancement. Given their non-toxic,
biocompatible, biodegradable characteristics, and their size-depen-
dent design for in vivo studies, they have been employed widely in
drug delivery,1,2 bio-sensing,3 and other biomedical applications such
as bio-separation4,5 and magnetic resonance imaging (MRI) contrast
enhancement.6–9 All of these scientific achievements have relied on the
nanoparticles’ multivalency, which helps to distinguish iron nano-
particles from other targeting agents. In fact, one functionalized
particle can carry multiple copies of an identical biological targeting
agent or a mixture of different targeting/imaging agents.
Experimental methods
Bovine serum albumin (BSA), folic acid, dicyclohexylcarbodiimide
(DCC), fluorescein isothiocyanate (FITC), Fluorescamine, and
reaction solvents were obtained from Sigma Chemical Company
(St Louis, MO) and used without further purification. Human
293 and A549 cells were obtained from the laboratories of Dr Vito
Quaranta and Dr Dennis Hallahan, respectively, at Vanderbilt
University. RPMI-1640 medium without folic acid was obtained
from Invitrogen.
At present, the amine group appears to be an ideal chemical moiety
for the functionalization of iron nanoparticles, this is due to its strong
nucleophilicity and compatibility with a wide range of available
amine activated biological materials. On the other hand, the func-
tionalization of nanoparticles with carboxylic acid groups enables the
Developing superparamagnetic iron oxide (SPIO) nanoparticles
Dextran-coated iron oxide nanoparticles were fabricated in our
laboratory using the reported protocol, but with some modifica-
tions.15 In brief, a saturated mixture of dextran and ferric chloride was
coalesced with a ferrous chloride solution at room temperature. This
was followed by elevating the pH to 10 via the dropwise addition of
an ammonium hydroxide solution. The resulting solution was stirred
aVanderbilt University, Institute of Imaging Science, 1161 21st Avenue
South, AA 1105 MCN, Nashville, TN, 37232-2310, USA. E-mail:
wellington.pham@vanderbilt.edu; Fax: +1 615 322-0734; Tel: +1 615
936-7621
bDepartment of Biomedical Engineering, VU Station B 351631, 5824
Stevenson Center, Nashville, TN, 37235, USA
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for several hours. The synthesized particles were separated from the
starting materials using deionized water, and then dialyzed into buffer
representing the normal physiological concentrations of sodium
citrate and sodium chloride. The concentration of iron in the solution
was determined by inductively coupled plasma mass spectrometry
(ICP-MS). To obtain the desired concentration of SPIO nano-
particles for analysis, a stock solution of SPIO particles was diluted
1 : 10 000 in 1% nitric acid solution. Each experiment was performed
in duplicate.
3-(Oxiran-2-yl)propan-1-aminium 4-methylbenzenesulfonate
A fresh solution of dimethyldioxirane was prepared as follows:
acetone (13.0 mL, 177 mmol), sodium bicarbonate (12.0 g,
143 mmol), and water (20 mL) were added to a 100 mL three-neck
round bottom flask, which was fitted with a solid addition funnel,
argon inlet and Vigreux column. Attached to the Vigreux column was
a 50 mL two-neck round bottom flask equipped with a dry-ice
condenser, which as then attached to a house vacuum line equipped
with an inline vacuum trap. The entire setup was purged of air using
a steady stream of argon. Oxoneꢀ (25.09 g, 41 mmol) was added
slowly to the reaction mixture via the solid addition funnel over
a period of 1.5 hours while stirring constantly and ensuring a steady
flow of argon. Once the Oxoneꢀ addition was complete, the entire
system was put under the house vacuum for a period of 1 hour.
Dimethyldioxirane in acetone (20 mL, 0.099 M) was trapped in the
two-neck round bottom flask, which was subsequently sealed and
stored in a freezer until needed.
Syntheses of an epoxide linker
Pent-4-en-1-amine
Under an atmosphere of argon, potassium phthalimide (17.21 g,
92.9 mmol) was combined with anhydrous DMF (180 mL). After-
wards, 5-bromopent-1-ene (10.0 mL, 84.4 mmol) was added slowly,
and the resulting mixture was heated to 60 ꢀC for 16 hours. The
reaction was then cooled to room temperature and added to a solu-
tion of 90% NaCl water (400 mL). The resulting solution was
extracted with diethyl ether (3 ꢁ 200 mL). The combined organic
extracts were washed with brine (1 ꢁ 200 mL), dried over magnesium
sulfate, filtered and concentrated via rotary evaporation to give a light
yellow oil. Prolonged high vacuum pumping yielded a semi-solid
(18.17 g, quant.), which was combined with ethanol (200 proof,
125 mL) in a 250 mL round-bottom flask equipped with a reflux
condenser. To the heavily stirring solution, hydrazine hydrate (64%
solution in water, 6.4 mL, 84.4 mmol) was added slowly. The mixture
was then heated to 60 ꢀC for 16 hours, during which time the reaction
mixture formed a large amount of white precipitant. Concentrated
hydrochloric acid (30 mL) was added slowly after cooling the reac-
tion to room temperature. The reaction was heated to reflux for
3 hours and then cooled to room temperature, after which the solids
were filtered out and washed with ethanol (200 proof, 100 mL) and
dichloromethane (50 mL). The solvent was removed by rotary
evaporation to reveal a white solid. This solid was then taken up in
water (80 mL) and made basic, to litmus, by the addition of potas-
sium hydroxide pellets. The resulting solution was extracted with
diethyl ether (4 ꢁ 50 mL). The combined extracts were concentrated
To a dry 25 mL round bottom flask were added pent-4-en-1-
aminium 4-methylbenzenesulfonate (0.103 g, 0.40 mmol) and dry
ꢀ
CH2Cl2 (3.0 mL), which was then cooled to 0 C under an atmo-
sphere of argon. The freshly prepared dimethyldioxirane solution
(8.0 mL, 0.84 mmol) was addedꢀ slowly, and the resultant clear
reaction mixture was stirred at 0 C for a period of 3 hours. Dry
diethyl ether (10 mL) was then added, which produced a white
precipitant. The precipitant was filtered out and rinsed with addi-
tional cold diethyl ether (10 mL) then dried under high vacuum to
afford the final product as a white solid (0.109 mg, quant.). 1H NMR
(300 MHz, CDCl3): d 7.72 (AA0XX0, J ¼ 8.0 Hz, 2H), 7.64 (bs, 3H),
7.14 (AA0XX0, J ¼ 7.9 Hz, 2H), 2.83 (t, J ¼ 7.5 Hz, 2H), 2.71
(m, 1H), 2.59 (t, J ¼ 4.4 Hz, 1H), 2.31 (m, 4H), 1.65 (p, J ¼ 7.6 Hz,
2H), 1.49 (m, 1H), 1.30 (m, 1H).
Coupling epoxide linker with the nanoparticles
The nanoparticles were prepared as described above and resuspended
in PBS at 11 mg mLꢂ1 of iron. The epoxide linker was dissolved in
a stock solution at a concentration of 100 mg mLꢂ1. By controlling
the concentration of nanoparticles at 2 or 5 mg mLꢂ1, 500ꢁ and
2000ꢁ excess epoxide linkers (the number of molecules vs. particles)
were mixed, with the reaction performed overnight, at room
temperature, with gentle stirring. The free epoxide linker was
removed from the nanoparticles using Zeba Spin Columns (Thermo
Scientific, Rockford, IL) following the manufacturer’s recommended
procedure.
ꢀ
on a rotary evaporator at a temperature of 0 C to reveal a light
yellow oil, which was purified by short-path distillation. The product
1
distilled over at 95–100 ꢀC (1.463 g, 20%). H NMR (300 MHz,
CDCl3): d 5.84–5.71 (m, 1H), 5.02–4.89 (m, 2H), 4.75 (s, 2H), 2.66
(t, J ¼ 7.2 Hz, 2H), 2.06 (q, J ¼ 7.1 Hz, 2H), 1.50 (p, J ¼ 7.4 Hz, 2H).
HRMS-ESI m/z: 86.0962 (C5H11N + H requires 86.0964).
Pent-4-en-1-aminium 4-methylbenzenesulfonate
Quantification of epoxide linkers on the surface of the nanoparticles
To a solution of pent-4-en-1-amine (1.463 g, 17.2 mmol) in dry
diethyl ether (10.0 mL) at 0 ꢀC was slowly added a solution of
p-toluenesulfonic acid (3.277 g, 17.2 mmol) in dry diethyl ether
(12.0 mL). This produced a white precipitant, which was filtered out
and rinsed with additional cold diethyl ether (10 mL). Drying under
high vacuum yielded the title compound as a white solid (2.8894 g,
65%). 1H NMR (300 MHz, D2O): d 7.64 (AA0XX0, J ¼ 8.2 Hz, 2H),
7.28 (AA0XX0, J ¼ 8.1 Hz, 2H), 5.84–5.71 (m, 1H), 5.07–4.98
(m, 2H), 2.90 (t, J ¼ 7.6 Hz, 2H), 2.31 (s, 3H), 2.05 (q, J ¼ 7.1 Hz,
2H), 1.66 (p, J ¼ 7.5 Hz, 2H); 13C NMR (75 MHz, D2O) d 142.3,
139.4, 137.2, 129.3, 125.2, 115.6, 38.9, 29.6, 25.7, 20.4. HRMS-ESI
m/z: 258.1164 (C12H20NO3S requires 258.1164).
The copy numbers of coupled epoxide linkers on the surface of each
nanoparticle were determined by measuring the amine group with
Fluorescamine,16 and free epoxide linkers of a known amount were
used as the standard. Aliquots of the control (uncoupled) and
coupled nanoparticles were both pretreated with a 0.1 ꢁ 0.1 M
borate buffer (pH 9.5). A Fluorescamine stock solution was prepared
in acetone at 40 mg mLꢂ1. For each tested sample and blank, Flu-
orescamine was added to a final concentration of 1 mg mLꢂ1.
Following the addition of Fluorescamine, the samples were mixed
and allowed to stand for several minutes. Fluorescence was then
determined with a 400 nm excitation and a 460 nm emission.
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Conjugation of folate and/or FITC to epoxide linker-coupled
nanoparticles
values of 15, 55, 95, 135, 175 and 215 ms, FOV ¼ 40 ꢁ 40 mm2,
acquisition matrix ¼ 256 ꢁ 256 and slice thickness ¼ 1.0 mm).
NHS-folate and FITC were dissolved in dimethyl sulfoxide at a stock
concentration of 12.5 or 100 mg mLꢂ1, respectively. The pH of the
epoxide linker-coupled nanoparticles was exchanged to 9.5 borate
buffer using Zeba Spin Columns, and the iron concentration was
adjusted to 2 mg mLꢂ1. An aliquot of 1 mL (2mg) of nanoparticle
stock solution was double-labeled with FITC and folic acid. This was
accomplished by first adding 5 mL of FITC solution and stirring the
mixture at room temperature for 45 minutes prior to subsequent
addition of 80 mL of the NHS-folate solution. For the FITC or folic
acid only labeled nanoparticles, only one reactant was added. All the
reactions were performed overnight, after which the conjugated
nanoparticles were separated from the unreacted folic acid and FITC
by passing the reaction mixture through Zeba Spin Columns. The
suspension was concentrated to 5 mg mLꢂ1 iron via centricon for
future use.
Results and discussion
The incorporation of targeting active biomolecules onto the surface
of iron nanoparticles for imaging and therapy has proven to be very
useful in medical diagnostics.18 Indeed, the many useful applications
for functionalized iron nanoparticles, which have been conceived
over the years, have made novel chemical modification of the parti-
cles even more important. The iron nanoparticles used in our study
are encapsulated in dextran to enhance their size improving blood
pool distribution. This coating material can render them sufficiently
biocompatible and biodegradable to allow suitable in vivo imaging.
The hydroxyl groups on the surface of the particles, as a result of this
fabrication will be converted into stronger nucleophiles by employing
a novel bifunctional linker. Two criteria are taken into account in
designing this linker. First, it must possess a highly reactive functional
group to ensure reactions with hydroxyl groups. The electrophilic
group on the linker should react with the hydroxyl groups under the
simplest of conditions without the use of a catalyst. Second, the other
end of the linker must have a strong nucleophilic moiety to support
bioconjugation studies. Epoxide reactive agents are considered ideal
for this application given their stability and reactivity. Epoxides have
been used widely as a suitable functionality for the development of
affinity probes for protein labeling.19
Quantification of the conjugated FITC and folic acid
The copy number of FITC in each nanoparticle was estimated simply
by measuring the fluorescence strength of the labeled particles against
the free FITC with a known concentration. The extent of folate
conjugation was determined by spectrophotometric analysis at an
absorbance of 358 nm (3 ¼ 8643.5 Mꢂ1 cmꢂ1) based on a reported
procedure.17
As shown in Scheme 1, the synthesis of epoxy-amine linker 8
begins with the Gabriel synthesis, enabling for the conversion of
5-bromopent-1-ene 1 into the corresponding alkenylamine 4.
Protection of the newly formed amine group of compound 4 was
found to be necessary prior to the formation of an epoxide, due to the
fact that amines can be oxidized faster than olefins once exposed to
oxidizing agents. Furthermore, the final linker product must have the
amine groups blocked, since the existence of free amines would
undoubtedly cause an opening of the epoxide ring via nucleophilic
attack. Currently, the protection of amino groups in alkenylamines
can be achieved using well known chemical reagents, such as Fmoc,
Boc, or Cbz groups.20–22 However, deprotection of these groups
requires an extensive amount of work, which was not within the
scope of our objectives. Instead, we used a method reported by
Asensio et al. to protect compound 4 as an alkenylammonium
Cell culture and uptake studies
Human kidney derived 293 (folate receptor positive) and alveolar
basal epithelial derived A549 cells (lung carcinoma, folate receptor
negative) were cultured in an RPMI-1640 medium supplemented
with 10% fetal bovine serum, 100 U mLꢂ1 penicillin, and
100 mg mLꢂ1 streptomycin (Gibco BRL) under standard culture
conditions in a humidified incubator maintained at 5% CO2 at 37 ꢀC.
Cell uptake studies were performed using both cell lines with folate-
conjugated or folate-free nanoparticles. A total of 1 ꢁ 106 cells were
seeded in each well of a 6-well plate. The cells were then washed with
PBS, and 1.2 mL (2 mg mLꢂ1 of iron) of FITC-labeled folate-
conjugated or folate-free nanoparticle suspensions in folate-free
RPMI-1640 were added to each well. After 2 hours of incubation at
37 ꢀC, the cells were washed four times with pre-cooled PBS before
they were collected by scraping with a small amount of PBS. Half of
the cells (0.5 million) were pelleted and examined for fluorescence by
IVIS (IVIS 200, Xenogen). Images were acquired using the IVIS
imaging system with excitation set at 490 nm. Fluorescence was
collected through a long-pass filter and analyzed using Living
Imageꢀ software version 3.0. Half of the cells were resuspended with
PBS and mixed with an equal volume of 12% gelatin to produce the
middle layer for a phantom comprised of 3 layers, with 12% gelatin
on the top and bottom, and a 6% gelatin and cell mixture sandwiched
in the middle (all gelatin solutions were prepared in PBS).
Magnetic resonance imaging
Phantom MR imaging was performed on a 4.7 T Varian Inova
scanner. Suspended cells in 6% gelatin were scanned using a single
slice, T2-weighted, spin-echo sequence (TR ¼ 5 s, TE arrayed with
Scheme 1 Design and syntheses of a versatile epoxy amine linker for
functionalizing nanoparticles.
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Fig. 1 Functionalization of dextran-coated iron oxide nanoparticles
with an epoxy amine linker. The process resulted in approximately 40–60
amines on the surface (A). The multivalency of aminated nanoparticles
was demonstrated by incorporation of multiple numbers of different
active biomaterials on the surface (B).
Fig. 2 The specificity of the dual-labeled iron oxide nanoparticles in cell-
based optical imaging. Folate receptor positive (293) and negative cells
(A549) were incubated with the probe and imaged using white light
(A) and fluorescence imaging (B).
salt 5.23 In this approach, the protonation of amine groups is efficient
enough to prevent amine oxidation by a strong electrophilic
O-transfer reagent. Another unique design of this salt is that the
product is compatible with the aqueous condition of nanoparticles.
Surprisingly, when using conventional reagents, such as mCPBA
or hydrogen peroxide, the olefin oxidation did not progress smoothly.
It is noteworthy that Asensio also made similar observation in some
of the compounds. Although further investigation is needed, we
cannot exclude the possible interference of the bulky counterion
tosylate group. In light of this, we synthesized dioxirane 7 using
a previously reported procedure.24 Compound 7 converts olefin 5 into
the desired epoxide linker 8 with 90% yield.
To test the feasibility of functionalizing iron nanoparticles with the
newly developed linker, we treated iron nanoparticles with excess
epoxide linker 8 in PBS, at physiological buffer. The conjugation was
completed in an overnight reaction. To quantify the number of
amines on the surface of the particles, we used the spiro dye Fluo-
rescamine, which only reacts with free amines converting the non-
fluorescent Fluorescamine to a fluorescent product, the intensity of
which is proportional to the quantity of free amines. While we did
observe residual fluorescence in the control group, where we incu-
bated Flurosecamine with dextran-coated iron nanoparticles, this
value was accounted for by subtraction from all the measured
samples. This assay allowed us to estimate that approximately 40 to
60 copies of epoxy amine linkers were conjugated to each nano-
particle (Fig. 1A).
Fig. 3 The folate receptor positive cells (293) were incubated with either
aminated iron oxide nanoparticles (ꢂFolate) or the dual-labeled probe
(+Folate). The resulted cells were dispensed in gelatin-containing
phantom tubes for MR imaging.
FITC conjugation of epoxy-conjugated nanoparticles was evaluated
by spectrophotometric or fluorometry analysis. We found that about
20 folic acid molecules were conjugated onto the surface of one
nanoparticle. While there were approximately 3 FITC molecules per
nanoparticle, the dual-labeled probe remained stable for several
months while stored at 4 ꢀC (Fig. 1B).
The abundant number of reactive amino groups on each particle
renders the nanoparticles very useful for carrying a multiple copies of
homogenous or heterogeneous targeting molecules for imaging or
therapeutic applications. To address that notion, we labeled the
aminated nanoparticles with both folic acid and FITC, sequentially.
The former was activated as a succinimide ester while the latter has
isothiocyanate as an amine reactive group. The extent of folate and
To test the specificity of the probe, folate receptor positive (293)
and negative (A549) cells were incubated with the dual-labeled probe
for 2 h at 37 ꢀC. After incubation, both cells were washed extensively
with cold PBS to remove the unassociated nanoparticles. The cells
were then collected and confirmed by optical or MR imaging.
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which will facilitate the effort to integrate nanotechnology with
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Acknowledgements
The authors thank Michiyo Koyama for assistance in manuscript
preparation. This work is supported by a grant from NIA.
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