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Volume 9 | Number 42 | 2007

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ISSN 1463-9076 COVER ARTICLE

www.softmatter.org Shi et al.
Synthesis, characterization, and
Registered Charity Number 207890 intracellular uptake of carboxyl-
terminated poly(amidoamine)
dendrimer-stabilized iron oxide
nanoparticles
HOT ARTICLE
Sokolovski et al.
On the origin of the forward peak
and backward oscillations in the F +
H2(ν = 0) HF(ν’ = 2) + H reaction 1463-9076(2007)9:42;1-D
 PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

Synthesis, characterization, and intracellular uptake of

carboxyl-terminated poly(amidoamine) dendrimer-stabilized iron
oxide nanoparticles
Xiangyang Shi,* Thommey P. Thomas, Lukasz A. Myc, Alina Kotlyar and
James R. Baker, Jr*
Received 18th June 2007, Accepted 22nd August 2007
First published as an Advance Article on the web 7th September 2007
DOI: 10.1039/b709147h
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Published on 07 September 2007 on http://pubs.rsc.org | doi:10.1039/B709147H

We report the synthesis and characterization of a group of carboxyl-functionalized

poly(amidoamine) (PAMAM) dendrimers of generation 3 (G3) that were used for the
stabilization of superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs). Folic acid (FA)
molecules were conjugated onto the dendrimer surfaces in an attempt to achieve specific targeted
imaging of tumor cells that overexpress FA receptors using dendrimer-stabilized Fe3O4 NPs.
Fe3O4 NPs were synthesized using controlled co-precipitation of Fe(II) and Fe(III) ions and the
formed dendrimer-stabilized Fe3O4 NPs were characterized using transmission electron
microscopy (TEM) and polyacrylamide gel electrophoresis (PAGE). The intracellular uptake of
dendrimer-stabilized Fe3O4 NPs was tested in vitro using KB cells (a human epithelial carcinoma
cell line) that overexpress FA receptors. It appears that carboxyl-terminated PAMAM dendrimer-
stabilized Fe3O4 NPs can be uptaken by KB cells regardless of the repelling force between the
negatively charged cells and the negatively charged particles. In the presence of a large amount of
carboxyl terminal groups on the dendrimer surface, the receptor-mediated endocytosis of Fe3O4
NPs stabilized by FA-modified dendrimers was not facilitated. It implies that the surface charge
of dendrimer-stabilized magnetic iron oxide NPs in biological medium is an important factor
influencing their biological performance.

1. Introduction dendrimer surface, providing a unique platform for cancer

The development of a wide spectrum of nanoscale particles has
shown great promise for various biomedical and biotechnolo-
gical applications, such as cell type recognition, disease diag-
nosis, targeted therapeutics, intracellular imaging, and drug/
gene delivery.1–5 In particular, magnetic iron oxide nanopar-
ticles (NPs) have been used as contrast agents for magnetic
resonance (MR) imaging and as colloidal mediators for mag-
netic hyperthermia of cancer.6,7 For cancer biological applica-
tions, the surface of iron oxide NPs are often modified with
specific biological ligands in order for them to target cancerous
cells through the ligand–receptor interaction.8–15 Folic acid
(FA) has been shown as one of the most promising ligands for
targeting a range of human carcinomas including breast,
ovary, endometrium, kidney, lung, head and neck, brain,
and myeloid cancers that are known to express FA recep-
tors.16–18 There are a number of reports using FA-modified
iron oxide NPs to target cancer cells.19–22 However, the surface
modification of FA onto iron oxide NPs in these reports
involves multiple reactions with a general low yield.
Recent studies of dendrimer nanomedicine in our group
show that various targeting ligands, imaging dyes, and ther-
apeutic drug molecules can be covalently conjugated onto
Michigan Nanotechnology Institute for Medicine and Biological
Sciences, University of Michigan, Ann Arbor, MI 48109-0533, USA
cell targeting, imaging, and therapeutics.23–26 It is expected
that self-assembly of the dendrimeric nanodevices onto iron
oxide NPs could provide a means to modifying the NPs for
targeted imaging of cancer cells. This approach to functiona-
lizing iron oxide NPs is simple and can avoid complicated
synthesis and characterization of the intermediate products of
iron oxide NPs when reactions are performed on the NP
surfaces.
It is known that poly(amidoamine) (PAMAM) dendrimers
can be used to stabilize metal or other inorganic nanoparti-
cles.27,28 Literature data also show that carboxyl-terminated
dendrimers can be self-assembled onto iron oxide NP
surfaces.29,30 However, there are no literature reports related
to the self-assembly of targeting molecule-modified dendri-
mers with carboxyl termini onto iron oxide NP surfaces and
the relevant biological performance of the resultant NPs. In
this present study, we have synthesized and characterized a
group of carboxyl-terminated poly(amidoamine) (PAMAM)
dendrimers of generation 3 (G3) with FA ligands. These FA-
modified G3 PAMAM dendrimers were assembled onto iron
oxide NP surfaces for subsequent intracellular uptake studies.
To our knowledge, this is the first report related to the direct
self-assembly of dendrimeric nanodevices onto iron oxide NP
surfaces for cell biological studies. The results will provide a
basis for rational design of functional NPs for biomedical
applications.

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Scheme 1 Schematic representation of reactions used to functionalize G3 PAMAM dendrimers and the self-assembly of carboxyl-terminated G3
dendrimers onto the Fe3O4 NPs.
2. Experimental
2.1 Materials
Amine-terminated PAMAM dendrimers of generation 3
(G3
NH2) were purchased from Dendritech (Midland, MI).
Succinic anhydride, glycidol, FA, 1-ethyl-3-[3-dimethylamino-
propyl]carbodiimide hydrochloride (EDC), ferric chloride
hexahydrate (FeCl3
6H2O 4 99%), ferrous chloride tetrahy-
drate (FeCl2
4H2O 4 99%), sodium hydroxide (0.5 M in
water), hydrochloric acid (diluted in water from HCl 437%),
and all the other chemicals and solvents were purchased from
Aldrich and used as received. Water used in all the experi-
ments was purified using a Milli-Q Plus 185 water purification
system (Millipore, Bedford, MA) with resistivity higher than
18 MO cm. Cellulose dialysis membranes (MWCO = 1000)
were acquired from Fisher.
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2.2 Functionalization of G3 PAMAM dendrimers
The synthesis of functionalized G3 PAMAM dendrimers is
schematically presented in Scheme 1. The synthesis and char-
acterization procedures were followed from previous litera-
ture.13,31–33 For the synthesis of fully carboxylated G3
dendrimers (compound 1), 194.8 mg dry G3
NH2 was dis-
solved in 5 mL DMSO in a 25-mL round-bottom flask. To this
dendrimer solution 5 mL of DMSO solution containing 451.1
mg succinic anhydride (SAH) (molar ratio of SAH/–NH2 =
5 : 1) was added under vigorous stirring and reacted for 24 h.
The DMSO solution was dialyzed against water to remove the
excess SAH as well as the organic solvent. The aqueous
retentate was filtered through a membrane with a diameter
of 0.45 mm, and then lyophilized. Yield : 246.3 mg, 86.4%.
Compound 1 is denoted as G3
SAH.
Phys. Chem. Chem. Phys., 2007, 9, 5712–5720 | 5713
 The synthesis of FA-modified fully carboxylated G3 den-
drimers (compound 2) was followed from previous litera-
ture.24,25,34 Briefly, a 3-molar equivalent (FA/G3
NH2 =
3 : 1) of FA (19.2 mg, 43.5 mmol) in 5 mL DMSO was mixed
with a 5-mL DMSO solution containing EDC (376.6 mmol,
72.2 mg) and stirred for 3 h. This process activated the
g-carboxylic acid of FA for further reaction with G3
NH2.
The activated FA solution (10 mL in DMSO) was added to a
10-mL DMSO solution containing 100.16 mg G3
NH2 and
stirred for 3 d. Then, the FA-modified G3 dendrimers were
further carboxylated by adding a 10-mL DMSO solution
containing SAH (231.9 mg, 5 times molar excess of the G3
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terminal amine groups) as described above, followed by
Published on 07 September 2007 on http://pubs.rsc.org | doi:10.1039/B709147H
extensive dialysis against PBS buffer (3 times 4 liters) and
water (3 times 4 liters) for 3 d to remove the excess of reactants
and byproducts. The aqueous retentate was filtered through a
membrane with a diameter of 0.45 mm then lyophilized. Yield :
130.0 mg, 80.7%. Compound 2 is denoted as G3
FA3SAH.
Compound 3 was synthesized by partial hydroxylation of
G3
NH2, followed by FA conjugation, and the remaining
amine groups on G3 dendrimers were fully carboxylated. To
a 10-mL methanol solution containing 116.28 mg G3
NH2 in a
25-mL round-bottom flask, a 10-mL methanol solution of
18.7 mg glycidol (molar ratio of glycidol/G3
NH2 = 15 : 1)
was added dropwise while being stirred. The reaction was
stopped after 24 h. Then a 10-mL aliquot of the reaction
mixture was reacted with FA through EDC chemistry as
described above. A 3-molar equivalent (FA/G3 dendrimer =
3 : 1) of FA (11.1 mg, 25.2 mmol) in 3 mL DMSO was mixed
with a 2-mL DMSO solution containing EDC (126.0 mmol,
24.2 mg) and stirred for 3 h. The FA/EDC mixture solution was
then added to the 10-mL aliquot of dendrimer solution and
reacted for 3 d. Then, the FA-modified G3 dendrimers with
partial hydroxylation were further carboxylated by adding a
10-mL DMSO solution containing SAH (58.9 mg, 5 times
molar excess of the remaining G3 terminal amine groups) as
described above. The reaction mixture was diluted with H2O
and dialyzed against PBS buffer (3 times 4 liters), and water
(3 times 4 liters) for 3 d to remove byproducts and the excess
of reactants. The retentate was filtered through a membrane
with a diameter of 0.45 mm and lyophilized. Yield : 66.6 mg,
74.0%. Compound 3 is denoted as G3
(GlyOH)15FA3SAH.
Compound 4 was synthesized by carboxylation of the
remaining amine groups of G3 dendrimers that were partially
hydroxylated. Another 10-mL aliquot of the reaction mixture
containing partially hydroxylated G3 dendrimers was added
with a DMSO solution containing SAH with 4 times molar
excess of the remaining terminal amines of G3 dendrimer
(572.2 mmol, 57.2 mg) under vigorous stirring and reacted
for 24 h. The final mixture solution was dialyzed against water
(6 times 4 liters) for 3 d to remove the excess unreacted agents
as well as the organic solvent. The aqueous retentate was
filtered through a membrane with a diameter of 0.45 mm, then
lyophilized. Yield : 66.4 mg, 81.2%. Compound 4 is denoted
as G3
(GlyOH)15SAH.
2.3 Synthesis of Fe3O4 NPs
The synthesis of Fe3O4 NPs was followed from a previously
published procedure using controlled co-precipitation of Fe(II)
5714 | Phys. Chem. Chem. Phys., 2007, 9, 5712–5720
and Fe(III) ions.35 Briefly, 25 mL of 1 M FeCl3
6H2O, 0.5 M
FeCl2
4H2O, and 0.4 M HCl mixture solution was prepared
in water under vigorous stirring. The co-precipitation of Fe3O4
NPs was carried out in a three-neck round-bottom flask. The
above mixture solution was added to 250 mL of 0.5 M NaOH,
which was preheated to 80 1C before the co-precipitation
reaction. The reaction was protected under N2 atmosphere
and was vigorously stirred. Black powder was collected by
sedimentation with a help of an external magnetic field and
washed several times with water until stable ferrofluid was
obtained. Finally, the particles were redispersed in water.
2.4 Stabilization of Fe3O4 NPs using dendrimers
An aqueous solution (515 mL) containing 5 mg Fe3O4 NPs was
mixed with 1 mL functionalized G3 PAMAM dendrimers
(1 mg mL 1 in H2O), followed by shaking overnight. The
mixture solution was centrifuged (6000 RPM, 10 min) to
remove unadsorbed dendrimer materials. Then the particles
were redissolved into water and dispersed under intense
sonication. Five cycles of centrifugation/dispersion were ap-
plied to purify the dendrimer-stabilized Fe3O4 NPs. The Fe3O4
NPs were finally dissolved into water. For biological applica-
tions, the water was replaced with PBS buffer to store Fe3O4
NPs. The Fe concentration of the dendrimer-stabilized Fe3O4
NPs was determined using atomic absorption spectroscopy
(AA903, ARL). A defined volume of the NPs was digested in
1.0 M nitric acid before measurements.
1
2.5 H NMR spectroscopy
1
H NMR spectra of functionalized dendrimers were recorded
on a Bruker DRX 500 nuclear magnetic resonance spectro-
meter. Samples were dissolved in D2O before NMR measure-
ments.
2.6 UV-Vis spectroscopy
UV-Vis spectra were collected using a Lambda 20 UV-Vis
spectrometer. All dendrimer samples were dissolved in water
at a concentration of 1 mg mL 1 before measurement.
2.7 High-performance liquid chromatography (HPLC)
analysis
The reversed-phase (RP) HPLC system consisting of a System
GOLD 126 solvent module, a model 507 autosampler
equipped with a 100-mL loop, and a model 166 UV detector
(Beckman Coulter, Fullerton, CA) were used in this work. A
Jupiter C5 silica-based RP-HPLC column (250  4.6 mm,
300 Å) was purchased from Phenomenex (Torrance, CA). Two
Phenomenex Widepore C5 safety guards (4  3 mm) were also
installed ahead of the Jupiter column. The mobile phase was a
linear gradient beginning with 100 : 0 (v/v) water/acetonitrile
(ACN) at a flow rate of 1 mL min 1. Trifluoroacetic acid
(TFA) at 0.14 wt% concentration in water as well as in ACN
was used as a counterion to neutralize the dendrimer surface
charge. All the samples were dissolved into the aqueous mobile
phase (water containing 0.14% TFA) at the concentration of
1 mg mL 1. The detection of eluted samples was performed at
210 or 280 nm. The analysis was performed using Beckman’s
System GOLD Nouveau software.
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 2.8 Matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF) mass spectrometry
MALDI-TOF spectra were acquired using a Micromass
TofSpec-2E spectrometer. Linear mode was selected as the
operation mode. Ten mg mL 1 beta-indoleacrylic acid in
acetonitrile/H2O (v/v = 70 : 30) was used as the matrix.
One mg dendrimer samples were dissolved in 1 mL methanol
and then diluted 5 times by methanol to get the final concen-
tration of 0.2 mg mL 1. An equal volume of 0.2 mg mL 1
dendrimer solution and matrix solution were well mixed.
Then, one mL solution of the mixture was injected on the
spots of the target plate. Five picomole proteins of cyto-
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2.12 In vitro cellular uptake of dendrimer-stabilized Fe3O4
NPs
KB cells were plated on glass cover slips in 30-mm dishes in
FA-free medium. The cells were rinsed and added with 1 mL
serum-free RPMI and some of the dishes were pre-incubated
with 100 mM free FA for 30 min at 37 1C. The cells were then
incubated with 75 mg mL 1 dendrimer-stabilized Fe3O4 NPs at
37 1C for 2 h. The dishes were fixed with 2% p-formaldehyde
solution, stained with 2% potassium ferrocyanide in 5% HCl
(Perl’s reagent), and counter-stained with nuclear fast red
solution. The cover slips were mounted using Gel-Mountt,
and images were taken using an inverted light microscope.

chrome-C (12 359 g mol 1), myoglobin (16 951 g mol 1), and
trypsinogen (23 976 g mol 1) were used as external standards.
3. Results and discussion
2.9 Polyacrylamide gel electrophoresis (PAGE)
3.1 Synthesis and characterization of functionalized G3
Analysis of PAMAM dendrimers and dendrimer-stabilized PAMAM dendrimers
Fe3O4 NPs by PAGE was performed on a Micrograd vertical
It is generally known that carboxyl-terminated dendrimers can
electrophoresis system (Model FB-VE10-1, FisherBiotech,
be used to stabilize iron oxide NPs.29,30 In this study, we
Pittsburgh, PA).32,33 Precast 4–20% gradient express gels for
synthesized a group of functionalized G3 PAMAM dendri-
PAGE were obtained from ISC BioExpress (Kaysville, UT). A
mers with carboxyl terminal groups. These dendrimer carboxy-
commercial power supply (Model EC135-90; Thermo Electron
lates were used to stabilize superparamagnetic Fe3O4 NPs. A
Corporation, Milford, MA) was used. Tris-Glycine buffer (pH
variety of analytical techniques were utilized to characterize
= 8.3) was purchased from Invitrogen (Carlsbad, CA). It was
the dendrimer materials including HPLC, MALDI-TOF,
diluted 10 times to prepare the running buffer. PAGE separa-
UV-Vis spectrometry, and 1H NMR.
tions typically required 40–50 min at 200 V. In a typical run,
Fig. 1 shows the HPLC chromatograms of the functiona-
into each sample well 4 mL of a sample solution containing
lized G3 PAMAM dendrimers. In all cases, the dendrimer
2 mL 1 mg mL 1 PAMAM dendrimers and 2 mL bromophenol
materials are quite pure and homogeneous. A small shoulder
blue sucrose dye (50% sucrose, 1% bromophenol blue) was
peak at the right of the main peak for both G3
SAH and
injected. Developed gels were stained overnight with 0.025%
G3
FA3SAH is related to the dimer species, which originally
Comassie Blue R-250 in 40% methanol and 7% acetic
exists in the starting G3
NH2 dendrimer material. However,
acid aqueous solution. The gels were destained with an
for G3
(GlyOH)15SAH and G3
(GlyOH)15FA3SAH, there
aqueous solution containing 7% (v/v) acetic acid and 5% (v/v)
are no shoulder peaks. This is due to hydroxylation functio-
methanol.
nalization, which masks the dispersity of the dendrimer
materials as demonstrated previously in our group.13,32,36
2.10 Transmission electron microscopy (TEM) The longer elution time of both G3
FA3SAH and
TEM measurements were performed at 60 kV on a Philips
CM-100 microscope equipped with a Hamamatsu Digital
Camera ORCA-HR operated using AMT software (Advanced
Microscopy Techniques Corp, Danver, MA). High-resolution
TEM images were collected using a JEOL 3011 electron
microscope at 300 kV. TEM samples were prepared by
deposition of a diluted particle suspension (5 mL) onto a
carbon-coated copper grid and air-dried before the measure-
ment.

2.11 Cytotoxicity
KB cells (a human epithelial carcinoma cell line) maintained in
FA-free medium were incubated with dendrimer-stabilized
Fe3O4 NPs at 37 1C for 24 h, and the NPs were removed.
The cells were allowed to grow for another 4 d, and the cell
proliferation was determined by an XTT assay (Roche Diag-
nostic, cat. No. 1465 015) under the manufacturer’s instruc- Fig. 1 HPLC chromatograms of the functionalized G3
SAH (1),
tions. Absorbance measurements were performed using a G3
FA3SAH (2), G3
(GlyOH)15SAH (3), and G3
(GlyOH)15-
Spectra Max 340 ELISA reader (Molecular Devices, Sunny- FA3SAH (4) PAMAM dendrimers. The gradient used was 0–80%
vale, CA) at 492 nm. ACN (balance water) within 30 min. Injection : 30 mL.

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Fig. 2 MALDI-TOF mass spectra of G3 PAMAM derivatives of
G3
SAH (1), G3
FA3SAH (2), G3
(GlyOH)15SAH (3), and
G3
(GlyOH)15FA3SAH (4). Lines in the mass spectra indicate the
collected molar mass values.
G3
(GlyOH)15FA3SAH (as compared with G3
FA3SAH and
G3
(GlyOH)15SAH) might be due to the FA conjugation,
which increases the hydrophobicity of the dendrimer materi-
als. The molecular weights of the functionalized G3 PAMAMs
were measured using MALDI-TOF mass spectrometry (Fig.
2). It indicates that, after the surface functionalization, the
molar mass of dendrimers increases. By using the MALDI-
TOF molar mass data of G3
NH2 (Mw = 5732) and partially
hydroxylated G3 dendrimers (compound 5 in Scheme 1,
denoted as G3
(GlyOH)15NH2) (Mw = 6922), the number
of glycidol hydroxyl groups of G3
(GlyOH)15NH2 dendri-
mers can be calculated to be 16.1, which is close to the
calculated stoichiometry of 15. The number of FA molecules
conjugated onto the dendrimer surface can be estimated by
comparing the UV-Vis absorbance of G3
FA3SAH and
G3
(GlyOH)15FA3SAH (Fig. 3) with the standard calibration
curve of free FA molecules at the characteristic FA absorption
wavelength of 280 nm. Based on the molecular weight of
G3
FA3SAH and G3
(GlyOH)15FA3SAH dendrimers mea-
sured by MALDI-TOF, the number of FA molecules con-
jugated onto the dendrimer surface is 3.6, which is close to the
initial feed ratio.
Fig. 3 UV-Vis spectra of the G3
FA3SAH (a) and
G3
(GlyOH)15FA3SAH (b) dendrimers (1 mg mL 1 in H2O).
5716 | Phys. Chem. Chem. Phys., 2007, 9, 5712–5720
1
H NMR spectrometry (Fig. 4) further confirmed the
structures of the functionalized G3 dendrimers. The NMR
peak assignment (bottom panel of Fig. 4) confirms the forma-
tion of PAMAM succinamic acids for all four compounds as
well as the partial hydroxylation reaction for both
G3
(GlyOH)15FA3SAH and G3
(GlyOH)15SAH; this is con-
sistent with our previous report.32,37 In Fig. 4b and d, the
multiple NMR peaks in the aromatic region (as magnified in
the insets) indicate the presence of FA moieties, in agreement
with previous literature.25
3.2 Synthesis and characterization of Fe3O4 NPs
The Fe3O4 NPs were synthesized by controlled co-precipita-
tion of Fe(II) and Fe(III) ions under a basic condition at an
elevated temperature. The Fe3O4 NPs formed are water-
soluble and have a diameter of 8.4  1.4 nm with narrow
dispersity (Fig. 5). Both high-resolution TEM images and
selected area electron diffraction (SAED) patterns of the
Fe3O4 NPs (Fig. 6) show that the synthesized Fe3O4 NPs are
polycrystalline.
3.3 Dendrimer-stabilized Fe3O4 NPs
Four different G3 PAMAM dendrimers were utilized to
stabilize Fe3O4 NPs through electrostatic assembly (Scheme
1). The positive surface potential of the synthesized Fe3O4 NPs
(+42.02 mV), which was verified by zeta potential measure-
ments, ensures the successful electrostatic assembly of car-
boxyl-terminated G3 dendrimers onto the Fe3O4 NP surfaces.
It is found that G3
SAH, G3
FA3SAH, G3
(GlyOH)15SAH,
and G3
(GlyOH)15FA3SAH work very well to stabilize the
Fe3O4 NPs, as no aggregation of Fe3O4 NPs appears in all
cases after stabilization for at least 3 months. TEM images
show that Fe3O4 NPs after dendrimer stabilization display
similar morphology to the ones without dendrimer modifica-
tion (images not shown). The dendrimer-stabilized Fe3O4 NPs
were then characterized using PAGE. Our previous study has
shown that PAGE is a powerful technique to characterize
dendrimer-stabilized nanoparticles.28 Fig. 7 shows the PAGE
electropherograms of both G3 PAMAM dendrimers and G3
dendrimer-stabilized Fe3O4 NPs with different concentrations.
It is clear that G3
(GlyOH)15SAH and G3
(GlyOH)15
FA3SAH display lower electrophoretic mobility than
G3
SAH and G3
FA3SAH because of their partially hydro-
xylated surfaces. For G3
SAH- and G3
FA3SAH-stabilized
Fe3O4 NPs, higher concentration injection generates a much
denser stain with Coomassie Blue, suggesting that more Fe3O4
NPs migrate under the electric field. It indicates that the
coating of dendrimers onto Fe3O4 NP surfaces is stable.
Fe3O4 NPs without dendrimer coating migrate reversely be-
cause of their positive surface potential.
3.4 Biological evaluation of dendrimer-stabilized Fe3O4 NPs
Before the study of the intracellular uptake of dendrimer-
stabilized Fe3O4 NPs, we first detect the cytotoxicity of all
dendrimer-stabilized Fe3O4 NPs. The XTT assay data (not
shown) verify that all dendrimer-stabilized Fe3O4 NPs do not
display cytotoxicity to KB cells in the concentration range of
0–80 mg mL 1.
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Fig. 4 1H NMR spectra of G3
SAH (a), G3
FA3SAH (b), G3
(GlyOH)15SAH (c), and G3
(GlyOH)15FA3SAH (d) dendrimers. The bottom
panel shows the assignment of the NMR peaks.

Fig. 5 TEM micrograph (a) and the size distribution histogram (b) of the synthesized Fe3O4 nanocrystals.

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Fig. 6 A high-resolution TEM image of the synthesized Fe3O4 NPs (a) and the selected area electron diffraction pattern of the same NPs (b).
In general, positively charged NPs always bind to cells
through electrostatic interaction with the negatively charged
cell membranes.38 We expect that the interaction between
negatively charged NPs and cell membranes might be differ-
ent. Interestingly, intracellular uptake of Fe3O4 NPs shows
that both G3
SAH- and G3
FA3SAH-stabilized Fe3O4 NPs
bind to the KB cells that overexpress FA receptors (Fig. 8a
and 8c). The dark (blue in a colored image) stain using Perl’s
reagent indicates the presence of Fe3O4 NPs. Control cells
without treatment of dendrimer-stabilized Fe3O4 NPs do not
display dark stains (Fig. 8e). In the presence of free FA
molecules, there is less binding for both NPs and more clusters
formation of the particles (Fig. 8b and 8d). For
G3
(GlyOH)15SAH- and G3
(GlyOH)15FA3SAH-stabilized
Fe3O4 NPs, we found a similar cellular uptake.
G3
(GlyOH)15FA3SAH-stabilized Fe3O4 NPs essentially do
not show more significant binding with KB cells, as compared
to G3
(GlyOH)15SAH-stabilized Fe3O4 NPs (images not
shown). It indicates that the negatively charged carboxyl-
terminated PAMAM dendrimer-stabilized Fe3O4 NPs can be
Fig. 7 PAGE electropherograms of G3 dendrimers and the G3
dendrimer-stabilized Fe3O4 NPs with different concentrations. Lanes
1–10 are (1) G3
SAH (4 mL), (2) G3
FA3SAH (4 mL), (3)
G3
(GlyOH)15SAH (4 mL), (4) G3
(GlyOH)15FA3SAH (4 mL), (5)
blank, (6) Fe3O4/G3
SAH (2 mg mL 1, 8 mL), (7) Fe3O4/G3
SAH (2
mg mL 1, 4 mL), (8) Fe3O4/G3
FA3SAH (5 mg mL 1, 8 mL), (9)
Fe3O4/G3
FA3SAH (1 mg mL 1, 8 mL), and (10) Fe3O4 (5 mg mL 1,
8 mL). The injection volume in each lane contains equal volumes of the
dendrimers (1 mg mL 1), dendrimer-stabilized Fe3O4 NPs (with
different concentrations) and standard bromophenol blue sucrose
dye solution.
5718 | Phys. Chem. Chem. Phys., 2007, 9, 5712–5720
uptaken by KB cells with a negative surface charge, although
the repelling force may lead to a certain degree of the cluster
formation of NPs on the cell surfaces. The cellular uptake of
carboxyl-terminated-dendrimer-stabilized Fe3O4 NPs may
occur through two distinct mechanisms : both phagocytosis
and diffusion via cell walls may take place. This is in agreement
with our previous report in the case of carboxyl-terminated-
dendrimer-stabilized Ag NPs.39
Our intracellular uptake data also suggest that
G3
FA3SAH- and G3
(GlyOH)15FA3SAH-stabilized Fe3O4
NPs do not show high affinity binding with KB cells over-
expressing FA receptors through ligand–receptor interaction,
as compared with G3
SAH- and G3
(GlyOH)15SAH-stabi-
lized Fe3O4 NPs, respectively. It is worthwhile to note that the
density of FA moiety on each Fe3O4 NP is much higher than
that of a single dendrimer agent which displays targeting
specificity, because each Fe3O4 NP is surrounded/protected
with multiple dendrimer molecules. The surface curvature of
dendrimer may not influence the binding of FA moiety with
the FA receptors on the cell surface, because the morphology
of dendrimers deposited on a solid particle surface displays a
pancake shape regardless of the dendrimer generation.40,41
Therefore, both the FA moiety density and dendrimer surface
curvature are not factors influencing the specific intracellular
uptake. We think that the non-specific uptake may be due to
the presence of a large amount of carboxyl groups on the
dendrimer surface. A previous report42 related to targeted
uptake of G5 dendrimer-RGD-4C peptide conjugates by
integrin receptor expressing cells show that 75 mol.% acetyla-
tion of the total amine groups on G5 dendrimers is essential to
maintain the targeting efficacy of the dendrimer device. In
other words, 25 mol.% carboxylation of the total amine
groups on G5 dendrimers does not affect the targeting speci-
ficity of dendrimer nanodevices. It indicates that the density of
carboxyl groups of dendrimers may play an important role for
the specific targeting of tumor cells through ligand–receptor
interaction.
In general, for tumor cell targeting studies, the surface
charge of particles should be ideally maintained to neutral
or close to neutral.23–26 As is generally known, positively
charged NPs can always be uptaken by cells through electro-
static interaction with the negatively charged cells. However,
the neutralized PAMAM dendrimers (e.g., acetylated
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c the Owner Societies 2007
Downloaded by State University of New York at Albany on 10/04/2013 07:01:28.
Published on 07 September 2007 on http://pubs.rsc.org | doi:10.1039/B709147H
Fig. 8 Intracellular uptake of G3
SAH- (a) and G3
FA3SAH- (c) stabilized Fe3O4 NPs. (b) and (d) panels show the binding of G3
SAH- and
G3
FA3SAH-stabilized Fe3O4 NPs with the preincubation of free FA molecules, respectively. Panel (e) shows the morphology of control cells
without treatment.
dendrimers) may lack stabilization force in the case of the
stabilization of Fe3O4 NPs. Therefore, alternative approaches
should be developed to maintain the close to neutral charge of
dendrimer-stabilized Fe3O4 NPs for targeted imaging of
tumors. In another of our studies,43 we coated Fe3O4 NPs
with a bilayer of polystyrene sulfonate sodium salt (PSS) and
FA-modified amine-terminated G5 dendrimer through elec-
trostatic layer-by-layer assembly, followed by acetylation of
the remaining amines of the assembled G5 dendrimers to
neutralize the surface charge of Fe3O4 NPs. The thus formed
NPs show specific intracellular uptake by FA receptor expres-
sing KB cells. This study further supports our above claim that
the surface curvature of dendrimers on the Fe3O4 NPs is not a
factor influencing their specific targeting to tumor cells
through ligand–receptor interaction. Our current study pro-
vides a clear experimental indication that Fe3O4 NPs stabilized
by dendrimers with a large amount of carboxyl groups on the
surface do not facilitate the specific binding of cancer cells
through ligand–receptor interaction even if the targeting
ligands are modified on the dendrimer surface. It indicates
that the dendrimer surface charge is a key factor affecting the
specific uptake of dendrimer-stabilized Fe3O4 NPs through
ligand–receptor interaction.
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c the Owner Societies 2007
4. Conclusions
We have synthesized and fully characterized a group of
carboxyl-terminated G3 PAMAM dendrimers that were used
to stabilize Fe3O4 NPs. The functionalized carboxyl-termi-
nated G3 PAMAM dendrimers were found to be able to
stabilize Fe3O4 NPs and be able to be uptaken by cells
regardless of the repelling force between the negatively
charged cell surfaces and the negatively charged particles.
The FA-modified Fe3O4 NPs lack binding specificity in com-
parison to the non-FA-modified Fe3O4 NPs, which is presum-
ably due to the large amount of dendrimer surface carboxyl
groups. We are currently developing various new approaches
to using dendrimer-functionalized Fe3O4 NPs with neutral or
close to neutral surface charges for targeted tumor imaging. In
addition, theoretical simulation approaches will also be em-
ployed to direct the fabrication of functionalized NPs with
desirable biological performance.
Acknowledgements
We thank Haiping Sun and Sasha Meshinchi for their assis-
tance with the TEM experiments and valuable discussions, and
Phys. Chem. Chem. Phys., 2007, 9, 5712–5720 | 5719
 Roger A. Pinto for his help with atomic absorption spectro-
scopy measurements. This project has been funded in whole or
in part by the National Institutes of Health (NIH) (under the
contract # NIH 1 RO1 EB002657, NOI-CO-97111, and NIH 1
R01 CA119409) and the Michigan Economic Development
Corporation-Life Sciences Corridor Fund (under award
GR-472).
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