Hyperbranched polyglycerol-grafted superparamagnetic iron oxide nanoparticles: Synthesis, characterization, functionalization, size separation, magnetic properties, and biological applications

Article abstract of DOI:10.1002/adfm.201201060

For biomedical application of nanoparticles, the surface chemical functionality is very important to impart additional functions, such as solubility and stability in a physiological environment, and targeting specificity as an imaging probe and a drug carrier. Although polyethylene glycol (PEG) has been used extensively, here, it is proposed that hyperbranched polyglycerol (PG) is a good or even better alternative to PEG. Superparamagnetic iron oxide nanoparticles (SPIONs) prepared using a polyol method are directly functionalized with PG through ringa opening polymerization of glycidol. The resulting SPIONa PG is highly soluble in pure water (>40 mg mL^-1) and in a phosphate buffer solution (>25 mg mL^-1). Such high solubility enables separation of SPIONa PG according to size using size exclusion chromatography (SEC). The sizea separated SPIONa PG shows a gradual increase in transverse relaxivity (r₂) with increasing particle size. For biological application, SPIONa PG is functionalized through multistep organic transformations (-OH → -OTs (tosylate) → -N₃ → -RGD) including click chemistry as a key step to impart targeting specificity by immobilization of cyclic RGD peptide (Arga Glya Aspa Da Tyra Lys) on the surface. The targeting effect is demonstrated by the cell experiments; SPIONa PGa RGD is taken up by the cells overexpressing α_vβ ₃-integrin such as U87MG and A549. Copyright

Full text of DOI:10.1002/adfm.201201060

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Hyperbranched Polyglycerol-Grafted Superparamagnetic
Iron Oxide Nanoparticles: Synthesis, Characterization,
Functionalization, Size Separation, Magnetic Properties,
and Biological Applications
Li Zhao, Tokuhiro Chano, Shigehiro Morikawa, Yukie Saito, Akihiko Shiino,
Sawako Shimizu, Takuro Maeda, Takayoshi Irie, Shuji Aonuma, Hidetoshi Okabe,
Takahide Kimura, Toshiro Inubushi, and Naoki Komatsu*
1. Introduction
For biomedical application of nanoparticles, the surface chemical func-
Good solubility of individualized nano-
tionality is very important to impart additional functions, such as solubility
particles in a physiological environment
and stability in a physiological environment, and targeting specificity as an
is vital for their biomedical application as
imaging probe and a drug carrier. Although polyethylene glycol (PEG) has
been used extensively, here, it is proposed that hyperbranched polyglycerol
(PG) is a good or even better alternative to PEG. Superparamagnetic iron
oxide nanoparticles (SPIONs) prepared using a polyol method are directly
functionalized with PG through ring-opening polymerization of glycidol. The
resulting SPION-PG is highly soluble in pure water (>40 mg mL−1 ) and in a
phosphate buffer solution (>25 mg mL−1 ). Such high solubility enables sepa-
ration of SPION-PG according to size using size exclusion chromatography
(SEC). The size-separated SPION-PG shows a gradual increase in transverse
relaxivity (r2) with increasing particle size. For biological application, SPION-
PG is functionalized through multistep organic transformations (–OH →
–OTs (tosylate) → –N3 → –RGD) including click chemistry as a key step to
impart targeting specificity by immobilization of cyclic RGD peptide (Arg-
Gly-Asp-D-Tyr-Lys) on the surface. The targeting effect is demonstrated by
the cell experiments; SPION-PG-RGD is taken up by the cells overexpressing
αvβ3-integrin such as U87MG and A549.
an imaging probe and a drug carrier. In
this context, extensive investigations have
been carried out for preparation of indi-
vidualized nanoparticles with high solu-
bility. Polyethylene glycol (PEG) is most
frequently used as dispersant due to their
hydrophilicity together with biocompatible
and anti-biofouling properties.[1–3] In our
experience of nanodiamond (ND), how-
ever, the aqueous solubility of PEG-func-
tionalized ND (ND-PEG) is not sufficient
for their biomedical applications.[4] In
order to impart better solubility to ND, we
added the hydroxyl branches to the PEG
chain in the molecular design and carried
out polyglycerol (PG) grafting through
ring-opening polymerization of glycidol
initiating from the surface of ND.[5–7]
As a result, PG-functionalized ND (ND-
PG) exhibited 400 times larger solubility
Dr. L. Zhao, S. Shimizu, T. Maeda, T. Irie,
Prof. T. Kimura, Prof. N. Komatsu
Department of Chemistry
Shiga University of Medical Science
Seta, Otsu 520-2192, Japan
Prof. Y. Saito
Graduate School of Agricultural and Life Sciences
The University of Tokyo
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
T. Irie, Prof. S. Aonuma
E-mail: nkomatsu@belle.shiga-med.ac.jp
Department of Applied Chemistry
Osaka Electro-Communication University
Neyagawa, Osaka 572-8530, Japan
Prof. T. Chano, S. Shimizu, Prof. H. Okabe
Department of Clinical Laboratory Medicine
Shiga University of Medical Science
Seta, Otsu 520-2192, Japan
Prof. S. Morikawa, Prof. A. Shiino, Prof. T. Inubushi
Biomedical MR Science Center
Shiga University of Medical Science
Seta, Otsu 520-2192, Japan
DOI: 10.1002/adfm.201201060
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in phosphate buffer saline (PBS) than ND-PEG because of
more hydrophilic nature of hydroxyl group than ether linkage
and much denser coverage of the ND surface with hyper-
branched PG than linear PEG.[8] We also found that the PG
grafting is effective in preparing stable aqueous hydrosol of
individualized nanoparticles, enabling us to separate the size
of the ND-PG by size-exclusion chromatography (SEC). PG is
reported to be as biocompatible as,[9] more resistant to protein
adsorption than,[10] and more amenable for further chemical
derivatization than PEG. PG can be concluded as better alter-
native to PEG in view of biomedical applications. In addition,
the PG functionalization is found to be applied to not only ND,
but also inorganic nanoparticle such as zinc oxide to prepare
stable hydrosol with good solubility in a physiological envi-
ronment.[11] We expect that this methodology can be applied
to a wide variety of nanomaterials to provide them with suf-
ficient properties for their biomedical applications, such as
aqueous solubility and stability in a solution, biocompatibility,
and protein-resistivity. In particular, this methodology is prom-
ising to give general solution towards the common problem of
nanoparticle agglomeration in a solution, which is an intrinsic
property of most nanoparticles.[12]
Superparamagnetic iron oxide nanoparticles (SPIONs) are
one of the most well-known nanoparticles used in biomed-
ical applications, especially as a magnetic resonance imaging
(MRI) probe for diagnosis, and as a drug carrier and a hyper-
thermic agent for therapy.[13–20] These applications frequently
require good solubility and high stability of the nanoparticle
in a physiological environment, and strict control of the size
and the surface chemical functionality.[21,22] In order to fulfill
these requirements, SPION has been wrapped with dispersant
polymers.[13,23] Most of the commercial SPIONs are coated
with dextran and its derivatives.[17] Although dextran-coating
imparts good aqueous solubility to SPION, this results in
increase of the particle size and broadening of the size distri-
bution compared to the size and size distribution of the indi-
vidual SPION. In order to prepare a stable aqueous solution
and regulate the size of SPION, ligands with high affinity to
the surface of SPION have been investigated to immobilize
hydrophilic polymers such as PEG on the surface.[7,13,23,24]
On the other hand, SPION prepared via polyol process was
reported to have intrinsic hydrophilicity due to the hydroxyl
groups on the surface.[25,26] This is in marked contrast to
SPION prepared by conventional coprecipitation[27] and high-
temperature decomposition in organic phase,[17,28] which is
hydrophobic.[19,26,29] Quite recently, the hydroxyl groups on the
hydrophilic SPION were proven to serve as the starting func-
tionality for multistep covalent transformations on the sur-
face.[30] This implies that we do not need anchoring ligands
to immobilize hydrophilic polymers on the surface of SPION
and that we can utilize the hydroxyl groups on the surface to
graft hydrophilic polymers directly.[7] Herein, we report on the
direct PG functionalization on the surface of SPION to impart
high solubility in a physiological medium and further cova-
lent chemical functionalization to add a targeting property for
cancer cell. We also realized chromatographic size separation
of the PG-functionalized SPION (SPION-PG)[1,8,31] and con-
firmed the relationship between the particle size and the mag-
netic properties.[1,32–34]
2. Results and Discussion
2.1. Synthesis and Characterization of SPION
SPION was synthesized by thermal decomposition of iron (III)
acetylacetonate in triethylene glycol (TREG) at high temperature
according to the reported procedure.[25,26] Powder X-ray diffraction
(XRD) reveals that the prepared sample is Fe3O4 (magnetite) with
a highly crystalline inverse spinel structure (Supporting Informa-
tion Figure S1). From the scanning transmission electron micro-
scopy (STEM) image shown in Figure 1a, the prepared SPION
is found to be spherical with average diameter of 8.8 2.2 nm.
The highly crystalline structure of the as-prepared SPION is also
confirmed by high-resolution transmission electron microscopy
(HRTEM); (311) crystallographic layers are clearly observed in
Figure 1b. However, the results of elemental analysis (Table 1)
and IR spectrum (Figure 2a) indicate that small portion of organic
component is included in the nanoparticles. C and H were
detected in the elemental analysis, and absorption of O–H and
C–H was observed in the IR spectrum. Taking into account these
results as well as the reaction conditions to prepare the SPION,
the organic contaminant in the as-prepared one should be TREG
and/or its derivatives.[25,26,30] In fact, the C:H ratio of the organic
contaminant (78:22) calculated from the result of the elemental
analysis (Table 1) is not so different from that of TREG (84:16)
calculated from the molecular formula and weight.
The ratio between the TREG and SPION can be estimated by
the results of elemental analysis and thermogravimetric anal-
ysis (TGA) shown in Table 1 and Figure 3, respectively. If the
weight (95.10 wt%) other than C and H in the elemental anal-
ysis of SPION is occupied by Fe from Fe3O4, and O from TREG
(molecular formula: C6H14O4) and Fe3O4, wt% of Fe and O is
calculated to be 66.37% and 28.73%, respectively. This weight
ratio of C (3.80%):Fe (66.37%):O (28.73%) is corresponding to
the following ratio in number of these atoms; C (6):Fe (22):O
(34), indicating that the ratio of TREG:Fe3O4 is calculated to be
about 1:7.4 in number and 1:11 in weight. Since the Fe3O4 core
consists of highly crystalline structure as mentioned above,
most of TREG molecules are considered to be adsorbed on and/
or included near the surface of the SPION. The weight reduc-
tion (8.9% at 450 °C) in TGA shown in Figure 3a is consistent
with the weight% of TREG (8%) calculated from the above
weight ratio (TREG:Fe3O4 = 1:11), supporting the existence of
TREG near surface region. Some TREGs are considered to be
just physisorbed on the surface of the iron oxide nanoparticle
through van der Waals interaction as reported by Santamaria
et al.[30] However, coordination bonding between iron in Fe3O4
and oxygen in TREG is also conceivable as reported by Maity
et al.,[35] sustaining TREG or its derivatives on the surface of
SPION more tightly. In fact, the weight of the as-prepared
SPION was continued to decrease in TGA (Figure 3a) even after
the temperature exceeded the boiling point of TREG (285 °C). In
addition, iron is hard Lewis acid to bear strong affinity toward
hard Lewis base such as oxygen. Therefore, we can conclude
that a significant number of hydroxyl groups are supposed to
stably exist in the surface region of the SPION and that the
hydroxyl groups, bearing increased nucleophilicity through
coordination bonding with iron,[35] may initiate PG grafting
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Figure 1. a) STEM and b) HRTEM images of as-synthesized SPION and STEM images of SPION-PG c) before chromatographic separation, d) fraction
1, e) fraction 2, and f) fraction 3 after chromatographic separation.
T a b le 1 . Elemental analyses of as-prepared and functionalized SPIONs.
polymerization of glycidol at the hydroxyl groups on the sur-
face of SPION as shown in Scheme 1. Under the same reaction
conditions as those in our previous papers,[8,11] we bath-soni-
cated and, then, heated the suspension of SPION in glycidol
without any additives such as acid and base. The SPION was
well dispersed in glycidol with the aid of bath sonication owing
to the hydroxyl groups on the surface, discussed above. After
washing and lyophilizing, blackish flocculent solid (52.3 mg)
was obtained from the starting SPION (30 mg).
C
H
N
[wt%]
[wt%]
[wt%]
SPION
3.80
30.60
24.51
1.10
5.83
4.90
0.00
0.00
1.11
SPION-PG
SPION-PG-N3
through ring-opening polymerization of glycidol on the surface
of SPION, as will be mentioned below.[8]
The obtained solid was characterized qualitatively by FTIR,
STEM, and dynamic light scattering (DLS), and quantitatively
by TGA and elemental analysis. Since the nanoparticle after the
reaction, shown in STEM image (Figure 1c), has the same shape
(sphere), average diameter, and standard deviation (SD) (8.8
2.3 nm) as that (8.8 2.2 nm) before the reaction (Figure 1a),
we conclude that the core is individual SPION. The PG grafting,
as shown in Scheme 1, was confirmed by large increase of the
absorption bands at 3400, 2900, and 1100 cm−1 corresponding
2.2. PG-Functionalization of SPION and Characterization
of SPION-PG
In order to increase the solubility of SPION sufficient for bio-
medical application, we intended to initiate the ring-opening
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diameter and the SD of the water-soluble
SPION-PG was determined to be 24.9 5.1 nm
on the basis of the number distribution by
DLS (Supporting Information Figure S2 and
Table 2). The difference between the hydrody-
namic diameter of SPION-PG by DLS (Sup-
porting Information Figure S2b) and the core
one by STEM image (Figure 1c) is considered
to correspond to the thickness of the PG layer
on the iron oxide core. The thickness of PG
of the SPION-PG in water, calculated to be
8.9 nm (Table 2), is similar to that of ND-PG
(11 nm).[8] Similar thickness of the PG coat-
ings on smaller core of SPION (8.8 nm) than
ND (30 nm) may realize much higher aqueous
solubility of the SPION-PG (>40 mg mL−1 )
than ND-PG (>20 mg mL−1 ), as will be dis-
cussed below (Figure 4) .
v C-O-C
v N-H
v O-H
v C=O
v C-H
f)
v N
3
e)
d)
v S=O
v C-C (aromatics)
c)
b)
a)
From the result of the elemental analysis
of SPION-PG shown in Table 1, we can also
estimate the weight ratio between the inner
core of SPION and the outer shell consisting
of TREG and PG. The weight of one spherical
Fe3O4 nanoparticle with average diameter of
8.8 nm is 1.8 ag, which is calculated from the
volume of the nanoparticle (357 nm3) and the
density of magnetite (5.1 g cm−3 ). The weight
of TREG in and/or on the nanoparticle is
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber ( cm-1)
Figure 2. FTIR spectra of a) as-synthesized SPION, b) free PG, c) SPION-PG, d) SPION-PG-
OTs, e) SPION-PG-N3, and f) SPION-PG-RGD. Arrows indicate new absorption bands after
each transformation.
caluculated to be 0.17 ag from the weight ratio between SPION
and TREG (11:1), discussed above. Since the weight (%) other
than C, H, and N in the elemental analysis, 63.57%, corre-
sponds to the weight (%) of Fe and O, the weight ratio of C:(Fe
+ O) is 30.60:63.57 in Table 1. C, Fe, and O in SPION-PG con-
sist of CTREG + CPG, FeSPION, and OSPION + OTREG + OPG, respec-
to O–H, C–H, and C–O–C stretchings, respectively, as in the
cases of ND- and ZnO-PG.[8,11] This is also supported by the fact
that the FTIR spectrum of SPION-PG (Figure 2c) is similar to
that of free PG (Figure 2b), which is prepared by ring-opening
polymerization of glycidol in the absence of SPION but oth-
erwise under the same conditions. The mean hydrodynamic
tively, where CTREG, CPG, FeSPION, OSPION
,
O
TREG, and OPG denote C in TREG and PG,
91.1 %
Fe in SPION, and O in SPION, TREG, and
PG, respectively. From the weight ratio of
(CTREG + CPG):(FeSPION + OSPION + OTREG
O
100
90
80
70
60
50
40
30
20
10
0
a)
+
PG) = 30.60:63.57, the weight of the PG layer
in one particle is calculated to be 2.9 ag (see
the Supporting Information). Based on the
above calculations, the weight of one SPION-
PG with the average size is determined to be
4.9 ag and the weight ratio of SPION core
(1.8 ag):PG (2.9 ag):TREG (0.17 ag) is 37:60:3.
The ratio of the inner core and the outer
shell (37:63) based on the result of the ele-
mental analysis is slightly different from
that determined by TGA (46:54) shown in
Figure 3b. This is probably because a part
of the organic component in the outer shell
was converted to the carbon materials such
as graphite and amorphous carbon at high
temperature under a nitrogen atmosphere
in TGA to increase the weight of nonvola-
tile component consisting mainly of SPION.
In fact, about 5 weight% remained at high
temperature in the TGA of free PG under
45.5 %
b)
5.0 %
c)
50 100 150 200 250 300 350 400 450 500 550 600
Temperature (oC)
Figure 3. TGA profiles of a) as-synthesized SPION, b) SPION-PG, and c) free PG under N2
atmosphere.
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carboxylates in SPION-PAA were converted
to carboxylic acids under acidic conditions to
facilitate the aggregation through hydrogen
bonding, the neutral hydroxyl groups in
SPION-PG are not affected by pH value in
the media.[23] Such non-ionic and highly
hydrophilic characteristics of PG may afford
a “stealth” effect similar to PEG to avoid non-
specific interaction with opsonin proteins
and uptake by the reticuloendothelial system
(RES), improving biocompatibility and blood
circulation times.[2,3,22,30,37] Owing to the firm
binding of SPION–TREG and TREG–PG
discussed above, these aqueous solutions of
Scheme 1. Synthesis of SPION-PG through ring-opening polymerization of glycidol.
nitrogen as shown in Figure 3c, while free PG was fully com-
busted below 600 °C under air.[8]
the SPION-PG were very stable without any aggregation; nei-
ther precipitation nor significant change in the hydrodynamic
diameter was observed for more than eight months. Such high
stability of non-aggregated, or individualized, form is also
important for biomedical application to avoid reduction of tar-
geting efficiency, uptake by RES, and risk of embolism.[22] Even
freeze-dried solid sample can be readily redispersed in water
without any insoluble precipitates. SPION-PG exhibits much
higher solubility than as-synthesized SPION[26] and most of the
The number of TREG and glycerol incorporated in one
SPION-PG can be estimated from the amounts of TREG (0.17 ag)
and PG (2.9 ag); 6.6 × 102 molecules of TREG and 2.4 × 104
units of glycerol are considered to consist of the outer shell. A
degree of polymerization in SPION-PG is compared with that
of ND-PG in Table 3.[8] In the number of glycidol grafted on
the surface of the core, ND with average diameter of 30 nm is
about 13 times larger than SPION with average diameter of
8.8 nm. When the number is divided by the surface area,
SPION and ND exhibit similar values as shown in Table 3.
This indicates that a degree of ring-opening polymerization is
considered to be roughly controlled by surface area of nanopar-
ticle. The larger PG/core weight ratio in SPION-PG implies the
better aqueous solubility of SPION-PG than that of ND-PG, as
will be discussed below.
2.3. Chromatographic Separation of Highly Soluble SPION-PG
SPION-PG was soluble in pure water, PBS, and a phosphate
buffer (20 mM, pH 7.0) containing Na2SO4 (100 mM) (a mobile
phase for chromatographic separation), and the solubility was
found to be more than 40, 25, and 20 mg mL−1 , respectively.
Since we only used limited amount of the SPION-PG to deter-
mine the above solubility, actual solubility is expected to be
larger than the above one. Under acidic conditions at a pH
value below 4, no precipitation was observed in the SPION-
PG solution of 20 mg mL−1 , which is in marked contrast with
the poly(acrylic acid)-coated SPION (SPION-PAA).[36] While
Figure 4. Photographs of a) an aqueous solution of SPION-PG (40 mg/
mL) in response to a permanent magnet and b) SPION-PG-RGD dis-
solved in PBS (1.0 mg/mL).
T a b le 2 . Structural and magnetic properties of SPION-PG before and after chromatographic separation.
Sample
Retention time
[min]
Core sizea)
[nm]
Surface areab)
[nm2]
Hydrodynamic sizec)
[nm]
Size differenced)
[nm]
Thickness of PGe)
[nm]
r2
[mM−1 s−1
]
before separation
fraction 1
–
243
327
260
191
86.30
91.97
86.91
77.91
8.8 2.3
10.2 2.7
9.1 1.9
7.8 1.7
24.1 4.4, 24.9 5.1f)
28.9 5.8
16.3 3.8, 17.8 4.6 f) 8.2 1.9, 8.9 2.3f)
21.0-24.0
24.0-26.0
26.0-29.0
18.7 5.1
15.4 4.1
11.6 3.4
9.4 2.6
7.7 2.0
5.8 1.7
fraction 2
24.5 4.5
fraction 3
19.4 3.8
a)Average core size of SPION-PG determined by more than 200 particles in the STEM images (Figure 1c–f); b) According to the equation of 4πr2, where r is average radius
of the core; c)Mean diameter of the number distribution determined by DLS in buffer, unless otherwise noted; d) Difference between core and hydrodynamic sizes; e) Half
of size difference; f)In Milli-Q water.
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T a b le 3 . Quantitative comparison of SPION-PG and ND-PG in one particle with average size.
Core size
[nm]
Core weighta)
[ag]
PG weight
[ag]
Weight ratio of PG/ Number of glycerol Surface area of core Unit number/surface
core
unit
[nm2]
area [nm−2
]
2.4 × 104
3.1 × 105
2.4 × 102
2.8 × 103
SPION-PG
ND-PG b)
8.8
30
1.8
50
2.9
38
1.6
100
0.76
110
a)According to the equation of 4/3πdr3, where r is average radius of the core, d is the density of nanoparticles; b)Ref. [8].
polymer-coated SPIONs. Strong hydrophilicity and superpara-
magnetism of SPION-PG are simultaneously demonstrated in
Figure 4. When an aqueous solution of SPION-PG (40 mg/mL)
was subjected to an external magnetic field, the magnetic nano-
particles together with the aqueous medium were attracted to
the magnet due to the superparamagnetism of SPION and
strong hydrophilicity of PG.
Such high solubility of SPION-PG in an aqueous solu-
tion enables chromatographic separation as in the case of
ND-PG.[1,8,22,31] A phosphate buffer (pH 7.0, 20 mM) con-
taining Na2SO4 (100 mM) was used as mobile phase (flow rate:
1.0 mL min−1 ), and the elution was monitored with UV light at
254 nm. The typical elution profiles of SPION-PG and free PG
are shown in Figure 5. The chromatographic peak at 21-29 min
corresponds to the elution of SPION-PG. A very weak peak at
35 min is attributed to free PG included in SPION-PG as an
impurity, because the retention time is in accordance with that
of the authentic free PG.[8] Three fractions were collected at the
retention time of 21–24, 24–26, and 26–29 min from the begin-
ning to the end of SPION-PG elution. These fractions were
analyzed by DLS to determine the mean hydrodynamic diame-
ters of the number distribution. The mean number diameter of
the core was determined by STEM after desalinization through
dialysis against Milli-Q water.
The results including SD are summarized in Table 2. As the
fraction proceeds from 1 to 3, the core and hydrodynamic dia-
meters as well as the SD decrease from 10.2 2.7 nm to 7.8
1.7 nm (Figure 1d–f) and from 28.9 5.8 nm to 19.4 3.8 nm
(Supporting Information Figure S2), respectively. Although
fraction 1 has wider size distribution in both core and hydrody-
namic sizes than the SPION-PG before separation, the size dis-
tributions in fractions 2 and 3 are narrower as suggested by the
SD. Post-synthesis SEC offers more precise way to control the
particle size. The similar and relatively small SDs in the hydro-
dynamic size of SPION-PG before and after separation (Table 2)
may prove high monodispersibility of SPION-PG. The size dif-
ference calculated from the core and hydrodynamic diameters
is corresponding to the thickness of the PG layer (Table 2). The
thickness and its SD also decrease as the core size decreases
in fraction 1–3. Interestingly, the SDs of the core sizes in frac-
tions 1–3 are almost the same as those of the thickness of PG
layer, indicating that the core size and thickness of SPION-PG
include similar degree of the distribution.
The hydrodynamic size (nm) of the SPION-PG and the
thickness (nm) of the PG layer in Table 2 are plotted against the
core size (nm) as shown in Figure 6. They linearly correlated
very well with more than 99.9% correlation coefficient. The fol-
lowing two equations are drawn from these plots in Figure 6:
(1)
dhydrodynamic = 4.0dcore−11.5
tPG = 1.5dcore−5.9
(2)
where dhydrodynamic, dcore, and tPG are the
hydrodynamic and core sizes (nm) of the
SPION-PG, and the thickness (nm) of the PG
layer, respectively. Since all the SPIONs pos-
sess the spherical shape as shown in Figure 1,
we can draw the strict linear correlation of
hydrodynamic size of the SPION-PG and
thickness of PG layer with the core size of the
nanoparticle shown in Figure 6 and the above
equations. This is not the case of ND-PG
because of the inhomogeneous shape of
ND.[8] These equations indicate quantitatively
that smaller core diameter of SPION-PG leads
to smaller hydrodynamic diameter of SPION-
PG and thinner PG layer under the same
reaction conditions of the PG functionaliza-
tion. As shown in Table 3, about 100 units of
glycerol should exist in unit area (1.0 nm2)
fraction 2
SPION-PG
24.0-26.0 min
free PG
fraction 3
26.0-29.0 min
fraction 1
21.0-24.0 min
0
5
10
15
20
25
30
35
40
45
50
on the surface of the core. Although 100
units of glycerol occupy similar volume on
the unit area of the SPION with smaller and
larger sizes, smaller size can accommodate
Retention time (min)
Figure 5. Chromatograms of the elution of SPION-PG and free PG.
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Figure 1a and Table 2) is similar to that of the
SPION reported by Cai et al. (particle size:
8
40
1.1 nm),[26] the similar r2 of the SPION
Hydrodynamic size
Thickness
35
30
25
20
15
10
5
and SPION-PG can be attributed to little or
no effect of the hydrophilic PG function-
ality on the r2. Although Bao et al. reported
large influence of PEG length on the r2 of
the SPION, the r2 was stable at the length
shorter and longer than the critical length of
the PEG, where the r2 was changed dramati-
cally.[32] Therefore, the TREG and PG on the
surface of the SPION might have similar
influence of long PEGs such as PEG 2000
and 5000 in the report, which exhibited sim-
ilar r2. On the basis of the discussion, the r2
decrease from fraction 1 to 3 shown in Table 1
can be ascribed to size decrease of the core
in SPION-PG, because the large magnetic
particles possess higher magnetic moments
and hence distort the magnetic field more
efficiently to enhance r2.[23,34,39] Size effect of
SPION on r2 can be concluded to exceed the
effect of the PG coating in the case of SPION-
PG, at least, in this range of the particle size.
0
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
Core size (nm)
Figure 6. Plots of hydrodynamic size of SPION-PG and thickness of PG layer against the core
size. Error bars correspond to standard deviation (SD). Actual values are shown in Table 2.
2.5. Further Functionalization of SPION-PG and the Targeting
Effect of SPION-PG-RGD
the same units of glycerol in a thinner space on the surface
than larger size because of the larger curvature. This may be
the reason why the thickness of the PG layer linearly corre-
lates with the core size of the SPION as shown in Figure 6 and
the Equation (2). We hope that these equations are applicable
to wider size range and more kinds of spherical nanoparticles
functionalized with PG.
The PG on the surface of SPION not only enhances
hydrophilicity to provide SPION with high aqueous solubility
and stability as described above, but also offers extensibility to
further functionalization to add more functions and properties
to SPION-PG. Therefore, we immobilized targeting function-
ality at the hydroxyl groups in the PG layer through stepwise
organic transformation shown in Scheme 2 and applied it to
cells to confirm the targeting efficiency of the further function-
alized SPION-PG.[31]
In order to introduce cyclic RGD peptide to target the αvβ3-
integrin expressed in some kinds of cancer cell, the hydroxyl
groups (–OH) in the SPION-PG were converted to tosylate
(SPION-PG-OTs), which is a good leaving group frequently
used in organic synthesis, and subsequently to azide (SPION-
PG-N3) through nucleophilic substitution of the tosylate with
sodium azide.[40] The SPION-PG-N3 was reacted with cyclic
RGD peptide having acetylene terminus through click chem-
istry,[16,23,41,42] giving SPION-PG-RGD (Scheme 2). These
organic transformations were confirmed qualitatively by FTIR
as shown in Figure 2 and quantitatively by elemental analysis
as shown in Table 1.
2.4. Size Dependence of MRI Relaxivity in SPION-PG
It has been reported that not only the size of SPION but also
the thickness of the surface coating affects the magnetic prop-
erties of hydrophilic SPIONs.[13,32,33,38,39] As mentioned above,
we separated SPION-PG by SEC and defined the core size and
the thickness of PG layer as summarized in Table 2. In order to
correlate the size parameters of the SPION-PG with the mag-
netic properties, MRI transverse relaxivity (r2) were determined
by the slope of the linear approximation based on the following
equation (Supporting Information Figure S3):
1/T2 = 1/T20 + r2[Fe]
(3)
where T2 and T0 are the observed transverse relaxation times
2
of SPION-PG and pure water, respectively, and [Fe] is the iron
concentration of the solutions. The r2 of as-synthesized SPION-
PG is found to be 86.30 Fe mM−1 s−1 as shown in Table 2,
which is similar to that of the SPION prepared in almost the
same process (82.68 Fe mM−1 s−1 ).[26] Since the SPION we
prepared was not dissolved well in water as in the case of San-
tamaria et al.,[30] but not in the case of Cai et al.,[26] we were
not able to measure MRI of as-synthesized SPION. Since the
core size of our SPION-PG (average diameter: 8.8 2.3 nm in
The absorption bands at 1600, 1350, and 1176 cm−1 in the
spectrum of SPION-PG-OTs (Figure 2d) are attributed to the
vibrational modes of the benzene ring, asymmetric and sym-
metric stretchings of S=O bonds, respectively. SPION-PG-N3
clearly shows characteristic absorption band of azide at 2100 cm−1
(Figure 2e). After the click reaction, the azide absorption band
was consumed thoroughly to give two new bands at 1660 and
1540 cm−1 attributed to C=O and N–H stretchings of amide
bonds in cyclic RGD peptides (Figure 2f).
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Scheme 2. Synthesis of SPION-PG-RGD from SPION-PG through multistep organic transformations.
The elemental analysis of SPION-PG-N3 demonstrates the
azide functionalization of SPION-PG by appearance of N con-
tent and decrease in C and H contents as compared with those
of SPION-PG (Table 1). The number of azide in one SPION-
PG-N3 particle and the ratio in numbers of azide and hydroxyl
group are calculated from the result of the elemental analysis.
As discussed above, the weight of one SPION-PG particle is
4.9 ag and, therefore, the weight of C is 1.5 ag due to the C
(wt%) of 30.60%. The number of C atom in one SPION-PG
is calculated to be 7.6 × 104, and the same number of C atom
should be included in SPION-PG-N3. Based on the weight%
of C and N (24.51% and 1.11%, respectively) in Table 1, the
number of azide in one particle is determined to be 9.8 × 102.
Since 2.4 × 104 units of glycerol are considered to consist of the
PG layer as discussed above, one hydroxyl group out of 24 is
converted to azide, which is considered to lead subsequently to
RGD quantitatively because of disappearance of absorption of
azide in FTIR shown in Figure 2f.
The resulting SPION-PG-RGD exhibited good solubility
and stability in PBS as shown in Figure 4b and was applied to
cell experiment to confirm the αvβ3-integrin targeted cell labe-
ling. SPION-PG and SPION-PG-RGD were incubated with fol-
lowing three kinds of cancer cells; HeLa, U87MG, and A549.
The uptake of the nanoparticles was confirmed histologically
by Prussian blue staining.[21] No noticeable color change is
observed in the cells treated with SPION-PG (upper pictures in
Figure 7), indicating that SPION-PG cannot internalize into the
cells through the cell membrane. In contrast, U87MG and A549
treated by SPION-PG-RGD were stained blue, while HeLa was
not stained (lower pictures in Figure 7). Since cyclic RGD pep-
tide is known to have affinity to αvβ3-integrin,[43,44] a degree of
blue staining may depend on number of the integrin expressed
in the cells. U87MG and A549 are well-known to overexpress
the integrin,[45,46] facilitating the internalization of SPION-
PG-RGD into the cells. On the other hand, HeLa is concluded
to have much less number of the integrin than U87MG and
A549.[47] In the stained cells shown in Figure 7, sporadic blue
spots were detected in the perinuclear cytoplasmic vesicles of
U87MG and A549 cells.[45,46] These results suggest that SPION-
PG-RGD was incorporated into the cells through receptor-medi-
ated endocytosis based on the affinity of cyclic RGD peptide to
the integrin.[43,44,47] The targeting efficacy of SPION-PG-RGD
demonstrated here together with high solubility and MRI relax-
ivities of SPION-PG described above indicate that SPIO-PG-
RGD possesses a great potential as an agent for in vivo cancer
imaging and therapy.
3. Conclusions
A PG coating provided SPION with excellent solubility and sta-
bility in a physiological medium as well as good extensibility
to add more functions such as targeting effect. Because PG
is also reported to be biocompatible[9] and resistant to protein
adsorption,[10] PG is considered as one of the ideal surface func-
tionality for nanoparticle in view of biomedical application. We
demonstrated direct PG grafting of SPION prepared by polyol
method and characterization of SPION and SPION-PG quali-
tatively using FTIR, STEM, and DLS, and quantitatively using
elemental analysis and TGA. From the quantitative discus-
sion, various structural characteristics of SPION-PG were dis-
closed such as the weights of the inner core and the outer shell,
and number of glycerol unit consisting of the PG layer. Since
SPION-PG exhibited high solubility even in a buffer solution,
SPION-PG was separated according to the size by chromatog-
raphy. The hydrodynamic size of SPION-PG and the thickness
of PG were found to correlate linearly with the core size, indi-
cating that the core size controls the thickness of the coating.
The core size also affected the magnetic properties of SPION-
PG, but the thickness of the PG layer was considered to cause
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Figure 7. Prussian blue stained images of cancer cells treated with SPION-PG (upper) and SPION-PG-RGD (lower).
a retention time of 15.4 min on an analytical HPLC, and used without
further purification. ESI-MS: m/z 672.40 for [M+H]+ (C30H41N9O9,
calculated molecular weight: 671.30) (Supporting Information
Figure S4).
less influence. In view of biomedical applications, the targeting
moiety was immobilized by utilizing good extensibility of PG;
the hydroxyl groups in PG was converted to azide through
multistep organic transformations, and RGD peptide was
conjugated covalently through click chemistry. The resulting
SPION-PG-RGD demonstrated targeting specificity towards
cancer cells overexpressing αvβ3-integrin such as A549 and
U87MG cancer cells. We believe that SPION-PG with targeting
functionality works as an imaging probe, a drug carrier, and a
hyperthermic agent in vivo, and makes a breakthrough in the
field of nanomedicine.
Preparation of SPION-PG: SPION was prepared according to the
published polyol method with slight modification.[26] Briefly, Fe(acac)3
(0.53 g, 1.5 mmol) and triethylene glycol (30 mL) were mixed with the
aid of bath sonication, then slowly heated to reflux over 3 h under an
argon atmosphere. After cooling down, ethyl acetate (20 mL) was added
to the blackish homogeneous suspension, which mixed by magnetic
stirring. The nanoparticles were collected by centrifugation (50 400 g,
20 min) and washed with ethyl acetate for 4 times, then dried in vacuo
to give a blackish solid (0.13 g). After a suspension of thus synthesized
SPION (30 mg) in glycidol (6.0 mL) was sonicated at 25 °C for 1 h,
the resulting dispersion was magnetically stirred at 140 °C under an
argon atmosphere for 20 h and then allowed to cool down to room
temperature. The resulting blackish gel was diluted with Milli-Q water
(20 mL) in an ultrasonic bath, and the precipitates were recovered
after ultracentrifugation at 543 200 g for 20 min. This washing process
was repeated four times to remove free PG. The washed sample was
lyophilized to give a blackish flocculent solid (52.3 mg).
The core sizes of the SPION before and after PG functionalization
were determined with STEM by measuring the sizes of more than 200
particles using Nano Measurer 1.2.5 (Jie Xu, Fudan University). The
mean hydrodynamic diameters of the number distribution in aqueous
solutions of the SPION-PG (Table 2) were determined by DLS on a
Nanotrac UPA-UT151 (Microtrac, Inc.).
Solubility of SPION-PG was determined as follows; an aliquot
(100 μL) of a solution of SPION-PG in Milli-Q water was dropped into a
tin boat (4 × 4 × 11 mm, Elementar Analysensysteme GmbH), thoroughly
dried at 60 °C overnight, and weighed the dried solid with a microbalance
(XP-6, Mettler-Toledo International Inc.). The procedure was repeated
three times and the average value was used as the solubility. Solubility
of a buffer solution of SPION-PG was determined in a similar way, after
it was dialyzed against Milli-Q water (Spectra/Pro dialysis membrane,
MWCO: 12–14 kDa).
4. Experimental Section
Chemicals: Chemicals were used as received, unless otherwise noted.
Iron (III) acetylacetonate (Fe(acac)3), N-hydroxysuccinimide (NHS),
and N,N-dicyclohexyl carbodiimide (DCC) were obtained from Nacalai
Tesque Inc. Fe(acac)3 was purified by recrystallization from water/
methanol before use. Glycidol purchased from Kanto Chemical Co.,
Ltd was dried over 4 Å molecular sieves prior to use. Propiolic acid was
purchased from TCI. cyclic RGD (Arg-Gly-Asp-D-Tyr-Lys) was purchased
from Peptides International, Inc. The mobile phase of the SEC was
prepared by diluting the commercial pH 7.0 phosphate buffer (100 mM,
Nacalai Tesque Inc.) five times followed by adding solid Na2SO4 to make
the concentration in the phosphate buffer to be 100 mM.
Synthesis of Succinimidyl-3-propiolate: This compound was synthesized
according to the reported procedure with minor modification.[48] DCC
(0.169 g, 3.0 mmol) in ethyl acetate (10 mL) was added dropwise
to a solution of propiolic acid (0.21 g, 3.0 mmol) and NHS (0.345 g,
3.0 mmol) in ethyl acetate (20 mL) in ice/water bath with vigorous
stirring. The reaction was kept at 4 °C for 4 h, and then warmed up to
room temperature and stirred overnight. After filtration, the filtrate was
concentrated to give yellow solid. The crude product was purified by
recrystallization from dichloromethane/hexane and dried in vacuo, giving
light yellow crystalline (0.36 g, 2.2 mmol) in 72% yield. mp: 89–91 °C, 1H
NMR (270 MHz, CDCl3, δ) 3.31 (s, 1H), 2.88 (s, 4H).
Synthesis of SPION-PG-OTs: SPION-PG (30 mg) was dissolved in an
aqueous solution of NaOH (0.25 M, 6.0 mL) by bath sonication and then
cooled down to 0 °C in an ice bath. p-Toluenesulfonyl chloride (30 mg,
0.16 mmol) was dissolved in DMF (6.0 mL) and added dropwise into
the mixture under rapid stirring. The solution was stirred at 0-5 °C for 3
h and at room temperature for 10 h. The resulting solid was collected by
ultracentrifugation and purified in DMF/water by repeated redispersion/
ultracentrifugation cycles.
Synthesis of RGD Propargyl Amide: Cyclic RGD peptide (10.0 mg, 16.2
μmol) and succinimidyl-3-propiolate (2.7mg, 16.2 μmol) were dissolved
in DMSO (1.0 mL) and stirred at room temperature for 48 h. After
evaporation of DMSO, the residue were dispersed in water and filtered
to remove insoluble impurities. The desired product was obtained with
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Synthesis of SPION-PG-N3: Sodium azide (20 mg, 0.31 mmol) in
water (2.0 mL) was added into SPION-PG-OTs (20 mg) in DMF (6.0 mL)
and stirred at 90 °C for 6 h under an argon atmosphere. After cooling
down, the product was collected by ultracentrifuge and purified in water
by repeated redispersion/ultracentrifugation cycles.
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DMSO (1.0 mL) was added to a solution of SPION-PG-N3 (12 mg)
in water (1.5 mL). Copper(II) sulfate pentahydrate (5.8 mg) in water
(0.5 mL) and sodium ascorbate (8.4 mg) in water (0.5 mL) were added
into the mixture with vigorous stirring. The resulting brown suspension
was bath sonicated for 5 min and stirred at room temperature for 36 h.
Diluted ammonia was dropped into the suspension to dissolve insoluble
copper salts, giving a clear brown solution. The solid was collected by
ultracentrifugation and purified in 1% ammonia by repeated washing/
ultracentrifugation. The purified sample was dialyzed against PBS to
replace the medium to a physiological solution for cell experiments.
Size Sorting of SPION-PG by SEC: Chromatographic size separation
of SPION-PG was conducted according to the procedures described
previously.[8] The following three columns were connected for SEC
separation of SPION-PG: Cosmosil CNT-2000, CNT-1000, and CNT-300
(diameter: 7.5 mm, length: 300 mm, Nacalai Tesque Inc.) with pore sizes
of 2000, 1000, and 300 Å, respectively. A phosphate buffer (20 mm, pH
7.0) containing Na2SO4 (100 mm) was used as the mobile phase with a
flow rate of 1.0 mL min−1 . After the injection of the solution of SPION-PG
in the buffer (5.0 mg mL−1 , 2.5 mL), elution was monitored with UV light
at 254 nm. Three fractions with 21–24, 24–26 and 26–29 min in retention
time were collected. The number-based mean hydrodynamic diameters
in these solutions (Table 2) were determined by DLS on a Nanotrac
UPA-UT151 (Microtrac, Inc.).
Measuring MRI Relaxivity of SPION-PG: Samples were desalinized
and then well dispersed in Milli-Q water with known Fe concentration
(determined by ICP-OES). Short NMR tubes containing diluted samples
with varying Fe concentration were fixed in a water bath at room
temperature. The magnetometric measurements were conducted on a
MRI system (Agilent, UNITY INOVA) with a 7-Tesla magnet (JASTEC)
and a magnetic field gradient coil (Magnex). MR images were obtained
by a spin echo sequence (reception time (TR) = 4000 ms, echo time (TE)
= 14, 18, 50, 80, 100, 120 ms, slice thickness = 2 mm, and field of view
= 40 × 40 mm2). Region-of-interest (ROI) was set on the tubes and the
signal intensities were measured for each concentration. The T2 values
were calculated by exponential fitting.
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This work was supported financially by Science and Technology
Incubation Program in Advanced Region (JST), Industrial Technology
Research Grant Program (NEDO), and Grant-in-Aid for Challenging
Exploratory Research (JSPS).
Received: April 17, 2012
Revised: June 23, 2012
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