Bovine serum albumin BSA and cleaved BSA
Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and
Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa
Bovine serum albumin (BSA) and cleaved-BSA conjugated ultrasmall
Gd2 O3 nanoparticles: Synthesis, characterization, and application to
MRI contrast agents
Md. Wasi Ahmad a , Cho Rong Kim a , Jong Su Baeck b , Yongmin Chang b,d,∗ ,
Tae Jeong Kim c,d , Ji Eun Bae d , Kwon Seok Chae d,e , Gang Ho Lee a,d,∗
a
Department of Chemistry, College of Natural Sciences, Kyungpook National University (KNU), Taegu 702-701, South Korea
Department of Molecular Medicine and Medical & Biological Engineering, School of Medicine, KNU, Taegu 702-701, South Korea
c
Department of Applied Chemistry, College of Engineering, KNU, Taegu 702-701, South Korea
d
Department of Nanoscience and Nanotechnology, KNU, Taegu 702-701, South Korea
e
Department of Biology Education, Teacher’s College, KNU, Taegu 702-701, South Korea
b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• BSA and C-BSA conjugated ultrasmall
•
•
•
•
Gd2 O3 nanoparticles were synthesized.
BSA can bind many ultrasmall Gd2 O3
nanoparticles whereas C-BSA cannot.
Large water proton relaxivities were
observed.
High contrast MR images in a mouse
liver after intravenous injection were
observed.
These nanoparticles are potential
MRI contrast agents.
a r t i c l e
i n f o
Article history:
Received 15 November 2013
Received in revised form 23 February 2014
Accepted 1 March 2014
Available online 12 March 2014
Keywords:
BSA
C-BSA
Ultrasmall Gd2 O3 nanoparticle
Nanoparticle carrier
MRI
Contrast agent
a b s t r a c t
Bovine serum albumin (BSA) (Mn = 66.5 kD, size = 14 × 4 × 4 nm) is an attractive biological molecule for
biomedical applications because of its water-solubility and bio-compatibility. It can also bind many
ultrasmall nanoparticles (NPs) as confirmed in this study. We synthesized polyethylene glycol diacid
(PEGD) coated ultrasmall Gd2 O3 nanoparticles (PEGD-GNPs, the core davg = 2.0 nm), which were then conjugated to BSA and cleaved-BSA (C-BSA) (i.e. BSA-PEGD-GNPs and C-BSA-PEGD-GNPs) through amide
bonding. Large relaxivities were observed in both aqueous sample solutions (r1 = 6.0 s−1 mM−1 and
r2 = 28.0 s−1 mM−1 for BSA-PEGD-GNPs and r1 = 7.6 s−1 mM−1 and r2 = 22.0 s−1 mM−1 for C-BSA-PEGDGNPs). Three tesla T2 magnetic resonance imaging (MRI) in a mouse after the injection of an aqueous
sample solution of BSA-PEGD-GNPs into a mouse tail vein revealed significant negative contrast enhancements. Large relaxivities and in vivo MR images prove that BSA-PEGD-GNPs and C-BSA-PEGD-GNPs are
potential MRI contrast agents.
© 2014 Elsevier B.V. All rights reserved.
Abbreviations: BSA, bovine serum albumin; C-BSA, cleaved BSA; PEGD, polyethylene glycol diacid; NP, nanoparticle; GNP, Gd2 O3 NP; BRB, Britton–Robinson buffer; PBS,
phosphate buffer solution; MRI, magnetic resonance imaging; NCT, neutron capture therapy; CT, X-ray computed tomography; DTPA, diethylenetriaminepentaacetic acid; PBS,
phosphate buffer solution; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GPC, gel permeation chromatograph; HVEM, high voltage
electron microscope; XRD, X-ray diffraction; ICPAES, inductively coupled plasma atomic emission spectrometer; FT-IR, Fourier transform-infrared; TGA, thermo-gravimetric
analyzer; DU145, human prostate cancer cell; NCTC1469, normal mouse hepatocyte cell.
∗ Corresponding authors. Tel.: +82 53 950 5340; fax: +82 53 950 6330.
E-mail addresses: ychang@knu.ac.kr (Y. Chang), ghlee@mail.knu.ac.kr (G.H. Lee).
http://dx.doi.org/10.1016/j.colsurfa.2014.03.011
0927-7757/© 2014 Elsevier B.V. All rights reserved.
68
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
1. Introduction
2.2. Synthesis of PEGD coated ultrasmall GNPs (PEGD-GNPs)
Biological molecules have attracted considerable interest for
application to nanomedicine because they are fully biocompatible
and water-soluble. Bovine serum albumin (BSA) is an important
carrier protein in blood plasma for several ions and molecules. It has
a large dimension (14 × 4 × 4 nm) and heavy mass (Mn = ∼66.5 kD).
Thus, it may also carry several ultrasmall nanoparticles (NPs) with
a smaller size and mass than BSA. Biological molecules have several advantages over small molecules and polymers for biomedical
applications. First, the water-solubility of surface-modified NPs
generally increases with increasing mass of the ligands [1,2] and
thus, biological molecules will provide enhanced water-solubility
for NPs. Second, the NPs conjugated to biological molecules can
remain in the blood for a longer duration than free NPs and
Gd-chelates, allowing longer imaging times (so called blood-pool
imaging agents) and a higher likelihood of delivering NPs to the
targeted areas in a body [3–6].
This study makes use of BSA and ultrasmall Gd2 O3 NPs (GNPs)
for magnetic resonance imaging (MRI). Ultrasmall GNPs have
shown longitudinal (r1 ) and transverse (r2 ) water proton relaxivities larger than those of Gd-chelates because of the dense
population of Gd(III) in NPs [7,8]. Therefore, BSA which could bind
several ultrasmall GNPs may be useful for MRI. Here, ultrasmall
GNPs can be also applied to X-ray computed tomography (CT)
as CT contrast agents and neutron capture therapy (NCT) as NCT
agents because Gd has a large X-ray attenuation power (∼2.5 times
stronger than commercial iodine CT contrast agents) [9–12] and a
very large neutron capture cross section (∼254,000 barns) [13–16].
This implies that BSA conjugated ultrasmall GNPs could be also
useful for CT and NCT.
Previous studies on MRI using BSA include BSA[Gd-chelates]n
and BSA-GNPs (d = 20–40 nm) [3–6,10,17]. Enhanced water proton
relaxivities have been observed in both systems after conjugation to BSA. In addition, the in vivo application of BSA[Gd-DTPA]n
(DTPA = diethylenetriaminepentaacetic acid) showed a longer circulation time in the blood than Gd-DTPA, providing longer MR
imaging times in brain tumors and blood vessels in rats [3,4].
This study examined BSA and cleaved BSA (C-BSA) (99.9%), triethylene glycol (99%), polyethylene glycol diacid (PEGD) (Mn = 600),
BSA (Mn = ∼66.5 kD), phosphate buffer solution (PBS) (pH = 7.2),
HCl (>99%), N-hydroxysuccinimide (NHS) (98%), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) (>97%), boric acid
(>98%), phosphoric acid (>97%), and acetic acid (98%) were purchased from Sigma-Aldrich and used as received. Triply distilled
water was used for both washing the products and preparing the
MRI sample solutions.
2.3. Synthesis of C-BSA
To prepare C-BSA, 10 mmol of l-serine and 10 mmol of lhistidine were dissolved in 300 mL of a Britton–Robinson buffer
(BRB) (40 mM phosphate, 40 mM acetate, and 40 mM borate)
(pH = 5–6) at 60 ◦ C and under atmospheric conditions (Scheme 1b)
[19]. Here, a pH of 5–6 of the buffer solution was achieved by adding
NaOH slowly to the original buffer solution. After magnetic stirring
for 30 min, 156 mg of BSA was added to the solution, and the solution was stirred magnetically for 36 h at 60 ◦ C. After the reaction
was complete, the water was evaporated. This cleavage reaction
was repeated 10 times to obtain enough C-BSA. The masses of CBSAs were characterized by gel permeation chromatography (GPC)
and the result is summarized in Supporting Information. Two major
C-BSAs with masses of 6.67 and 2.01 kD were observed from GPC
analysis. The C-BSA was used without further purification because
the water-soluble C-BSA could not be separated from other watersoluble reagents used in the reaction. On the other hand, the water
soluble reagents were later removed after conjugation of C-BSAs to
PEGD-GNPs through amide bonding.
2.4. Synthesis of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs
BSA-PEGD-GNPs and C-BSA-PEGD-GNPs were synthesized
using an EDC/NHS coupling method (Scheme 1c) [20,21]. In this
reaction amide bonds were formed between –COOH of PEGD-GNPs
and –NH2 of BSA (or C-BSA). The reaction was carried out at room
temperature and under atmospheric conditions. Solution pH was
fixed at 6.0 by adding 1 mM HCl to the original PBS with pH of 7.2.
5 mmol of EDC and 5 mmol of NHS were added to 30 mL of PBS
(pH = 6). After magnetic stirring for 30 min, the PEGD-GNPs were
added to the solution and the solution was stirred magnetically
for 2 h. 1.5 g of BSA (or C-BSA) was added to the solution at room
temperature and stirred for an additional 2 h. The product solution
was transferred to a 1 L beaker containing 500 mL of triply distilled
water. The resulting solution was stirred magnetically for 10 min
and stored for a week until BSA-PEGD-GNPs (or C-BSA-PEGD-GNPs)
settled in the bottom of the beaker. The supernatant was decanted
and the remaining sample solution was washed again with triply
distilled water. This procedure was repeated three times. During
this process, any water-soluble impurities such as solvent and any
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
69
Scheme 1. Syntheses of (a) the ultrasmall GNPs and PEGD-GNPs, (b) the C-BSA, and (c) the BSA-PEGD-GNPs and C-BSA-PEGD-GNPs.
other reagents used in synthesis of C-BSA, were removed from the
products. Portions of the samples were evaporated to a powder
form in air and the remaining portions were diluted with triply
distilled water to prepare aqueous sample solutions for the MRI
experiments.
2.5. General characterization
The particle diameters of the ultrasmall GNPs were measured
with a high voltage electron microscope (HVEM) (JEOL JEMARM1300S, 1.2 MeV acceleration voltage). A copper grid (PELCO
No. 160, TED PELLA, INC.) covered with an amorphous carbon
membrane was placed onto a filter paper and a sample solution diluted in triply distilled water was dropped onto the copper
grid using a micropipette (Eppendorf, 2–20 L). The copper grid
was dried in air for one hour to remove the solvent. The crystal structure of ultrasmall GNPs was examined using an X-ray
diffraction (XRD) spectrometer (Philips, X-PERT PRO MRD) with
unfiltered Cu-K␣ radiation of 1.54184 Å, scanning step of 0.033◦ ,
and scan range of 2 = 15–100◦ . The concentration of Gd in the sample solution was determined using an inductively coupled plasma
atomic emission spectrometer (ICPAES) (Thermo Jarrell Ash Co.,
IRIS/AP). The surface coating of the ultrasmall GNPs with PEGD
and conjugation of PEGD-GNPs to BSA (or C-BSA) were investigated
using a Fourier transform-infrared (FT-IR) absorption spectrometer (Mattson Instruments, Inc., Galaxy 7020A). The powder samples
were dried on a hot plate at 50 ◦ C in a hood for one week to
minimize the water content. To record the FT-IR absorption spectra (400–4000 cm−1 ), pellets of the powder samples in KBr were
prepared. The amount of surface coating was estimated with a
thermo-gravimetric analyzer (TGA) (TA Instruments, SDT-Q 600).
The TGA curves of the powder samples were recorded between
room temperature and 700 ◦ C while air flowed. The amounts of
PEGD, BSA, and C-BSA per GNP were estimated by recording the TGA
curves. Water desorption between room temperature and ∼110 ◦ C
was considered in these estimations.
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Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
2.6. Relaxivity and map image measurement
Both the longitudinal (T1 ) and transverse (T2 ) relaxation times
and longitudinal (R1 ) and transverse (R2 ) map images of the
aqueous sample solutions of BSA-PEGD-GNPs and C-BSA-PEGDGNPs were measured using a 1.5 T magnetic resonance imaging
(MRI) instrument (GE 1.5 T Signa Advantage, GE medical system)
equipped with a Knee coil (EXTREM). Aqueous sample solutions at
different Gd concentrations were prepared by diluting the original sample solutions with triply distilled water. Both map images
and relaxation times were measured using these solutions. The longitudinal (r1 ) and transverse (r2 ) water proton relaxivities were
estimated from the slopes in the plots of 1/T1 and 1/T2 versus
the Gd concentration, respectively. The typical parameters used to
measure the relaxation times and map images were as follows:
the external MR field (H) = 1.5 T, the temperature (T) = 22 ◦ C, the
number of acquisition (NEX) = 1, the field of view (FOV) = 16 cm,
the phase FOV = 1, the matrix size = 512 × 512, the slice thickness = 5 mm, the spacing gap = 0, the pixel bandwidth = 61.0547, the
repetition time (TR) = 2009 ms, and the echo time (TE) = 9 ms.
2.7. In vitro cytotoxicity measurement
The in vitro cytotoxicity of the aqueous sample solutions of
BSA-PEGD-GNPs and C-BSA-PEGD-GNPs was measured using both
human prostate cancer (DU145) and normal mouse hepatocyte
(NCTC1469) cells. A CellTiter-Glo Luminescent Cell Viability Assay
(Promega, WI, USA) was used to measure the cytotoxicity. In this
assay, the intracellular ATP was quantified using a luminometer
(Victor 3, Perkin Elmer). The cells were seeded onto a 24-well cell
culture plate and incubated for 24 h (5 × 104 cell density, 500 L
cells per well, 5% CO2 , 37 ◦ C). A series of test sample solutions (0,
10, 100, and 200 M) were prepared by diluting the original sample
solutions with a sterile phosphate buffer saline solution. ∼2 mL of
each test solution was added to the cell culture media. The treated
cell culture media were then incubated for 48 h. The cell viability of
each cell was determined and normalized with respect to that of the
control cell with 0.0 M Gd concentration. The measurements for all
test cells were repeated twice to obtain the average cell viabilities.
2.8. In vivo 3 T T2 MR image measurement
A 3 T MRI instrument (SIEMENS 3.0 T MAGNETOM Trio a Tim)
was used to measure the T2 spin echo (SE) images of a mouse.
The animal experiment in this study was carried out under the
permission and guidance of the KNU animal committee. An ICR
female mouse (ICR—Institute of Cancer Research, USA) with a
weight of ∼100 g was used for the MR image measurements.
The mouse was anesthetized by 1.5% isoflurane in oxygen. The
measurements were made before and after injecting the sample
solution into a mouse tail vein. The injection dose was ∼250 L
(∼0.1 mmol Gd kg−1 ). After the measurement, the mouse was
revived from anesthesia, placed into a cage, and given a free access
to both food and water. During the measurement, each mouse
was maintained at ∼37 ◦ C using a warm water blanket. The typical measurement parameters were as follows: the H = 3 T, the
Fig. 1. HVEM images of (a) and (b) ultrasmall GNPs at two different scales, (c) BSA-PEGD-GNPs, and (d) C-BSA-PEGD-GNPs.
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
71
3. Results and discussion
through hydrogen bonding [23–25]. The estimated cell constants
are a = 6.644 Å, b = 6.841 Å, c = 6.328 Å, and ˇ = 103.976◦ for TGAtreated BSA-PEGD-GNPs and a = 6.643 Å, b = 6.839 Å, c = 6.326 Å, and
ˇ = 104.001◦ for TGA-treated C-BSA-PEGD-GNPs which are consistent with the values reported in PCPDFWIN [26].
3.1. Particle diameter (d) and crystal structure of ultrasmall GNPs
3.2. Surface modification of ultrasmall GNPs
Fig. 1a–d shows HVEM images of the as-prepared ultrasmall GNPs, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs. The particle
diameters of the ultrasmall GNPs ranged from 1 to 3 nm with the
davg of 2.0 nm (Fig. 1a and b). The HVEM image of BSA-PEGD-GNPs
indicated that many ultrasmall PEGD-GNPs were conjugated to a
BSA (Fig. 1c). On the other hand, the HVEM image of C-BSA-PEGDGNPs showed that many C-BSAs were conjugated to each ultrasmall
PEGD-GNP (Fig. 1d). As will be discussed later, this is consistent
with the numbers of PEGD-GNPs conjugated to BSA and C-BSA
estimated from TGA analyses.
The XRD patterns of powder samples of both BSA-PEGD-GNPs
and C-BSA-PEGD-GNPs were measured before and after TGA and
are provided in Fig. 2. The XRD patterns of the as-prepared powder samples were broad, due likely to ultrasmall particle diameters
[22]. On the other hand, the XRD patterns of the TGA-treated powder samples revealed sharp peaks, corresponding to monoclinic
GdPO4 . All peaks after TGA analysis could be assigned to monoclinic
GdPO4 and the peak positions with sufficient intensities are marked
with ‘*’ in Fig. 2 and the Miller index (h k l) assignments of these
peaks are provided in Supporting information. The formation of
GdPO4 after TGA analysis is because the EDC/NHS coupling reaction
was carried out in PBS. That is, the PO4 3− ions were likely attached
to amine groups of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs
The surface coating of ultrasmall GNPs with PEGD followed by
conjugation to BSA or C-BSA was investigated by FT-IR absorption
spectroscopy. As mentioned previously, one group among the two
–COOH groups in each PEGD was conjugated to an ultrasmall GNP
and the other was left free for amide bonding to BSA or C-BSA.
These were confirmed from the two different C=O stretching
vibrations in the FT-IR absorption spectrum of the PEGD-GNPs
(Fig. 3a). The free –COOH was observed at 1730 cm−1 but the
–COOH bonded to GNPs, at 1620 cm−1 . The peaks at 2910 cm−1
(C–H stretch) and 1110 cm−1 (C–O stretch) also confirmed that the
PEGDs were bonded to ultrasmall GNPs. The peak at 3420 cm−1
in the PEGD-GNPs was assigned to the water –OH stretch. The
∼110 cm−1 red shift of the C=O stretch after bonding to GNPs
from that of the free –COOH had been observed in a range of
the metal oxide NPs coated with the ligands with –COOH groups
[22,27–30], supporting this result. The successful amide bond
formation between PEGD-GNPs and BSA (or C-BSA) was confirmed
from the disappearance of a free C=O stretch at 1730 cm−1 in both
BSA-PEGD-GNPs and C-BSA-PEGD-GNPs (Fig. 3b). Instead, the N–H
stretch at 3440 cm−1 (overlapped with water –OH stretch) and the
N–H bend at 1530 cm−1 were observed [31–33]. The reduced intensity in N–H bend in both BSA-PEGD-GNPs and C-BSA-PEGD-GNPs
was, however, observed owing to the amide bonding of amine
groups of BSA and C-BSA with PEGD-GNPs and the hydrogen
bonding of amine groups of BSA and C-BSA with PO4 3− ions.
T = 37 ◦ C, the NEX = 3–4, the FOV = 60 mm, the phase FOV = 30 mm,
the matrix size = 128 × 256, the slice thickness = 1 mm, the spacing
gap = 0.1 mm, the TR = 2690 ms, and the TE = 37 ms.
*
* : GdPO4
(a)
*
*
after TGA
*
*
*
*
*
*
*
* *
*
*
*
*
*
*
*
*
as prepared
20
40
60
Transmittance (%)
Intensity (Arb. Units)
(a)
PEGD-GNP
1730
3420 2910
2
*
1620
1110
3000
2000
1000
-1
Wavenumber (cm )
* : GdPO4
(b)
*
*
*
*
after TGA
*
**
*
*
*
*
*
*
*
*
*
*
*
as prepared
20
40
60
2
Fig. 2. XRD patterns of powder samples of (a) BSA-PEGD-GNPs and (b) C-BSA-PEGDGNPs before (i.e. as-prepared) and after TGA analysis. All peaks after TGA analysis
could be assigned to monoclinic GdPO4 and only the peak positions with sufficient intensities are marked with ‘*’ (Miller index (h k l) assignments are provided
in Supporting information).
Transmittance (%)
Intensity (Arb. Units)
4000
*
(b)
PEGD
BSA
BSA-PEGD-GNP
2910
1620 1530
C-BSA-PEGD-GNP
3440
4000
3000
2000
1000
-1
Wavenumber (cm )
Fig. 3. FT-IR absorption spectra of powder samples of (a) PEGD and PEGD-GNPs and
(b) BSA, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs.
72
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
100
30
Water desorption = 9.8%
(a)
(a)
28.0%
60
20
-1
40
10
-1
0
-1
r1 = 6.0 s mM
20
0
0
0.00
100 200 300 400 500 600 700
o
Temperature ( C)
100
Water desorption = 4.9%
0.25
0.50
0.75
Concentration Gd (mM)
1.00
6
(b)
80
5
47.4%
(b)
-1
-1
r2 = 22.0 s mM
4
-1
)
60
1/T (s
Weight (%)
-1
r2 = 28.0 s mM
-1
1/T (s )
Weight (%)
80
40
20
3
-1
-1
r1 = 7.6 s mM
2
1
0
0
100
(c)
100 200 300 400 500 600 700
o
Temperature ( C)
Weight (%)
61.7%
0.04 0.08 0.12 0.16
Concentration Gd (mM)
0.20
Fig. 5. Plots of 1/T1 and 1/T2 of the aqueous sample solutions of (a) BSA-PEGDGNPs and (b) C-BSA-PEGD-GNPs as a function of the Gd concentration. The slopes
correspond to the r1 and r2 values, respectively.
and 4.4 × 1.2 × 1.2, respectively, assuming a cube root dependence
of the size on the mass. Therefore, C-BSA is not large enough in
mass and size to bind many ultrasmall GNPs, which is similar to
the polymers.
40
20
0
0.00
Water desorption = 7.3%
80
60
0
0
100 200 300 400 500 600 700
o
Temperature ( C)
Fig. 4. TGA curves of powder samples of (a) PEGD-GNPs, (b) BSA-PEGD-GNPs, and
(c) C-BSA-PEGD-GNPs.
To determine the amounts of BSA in the BSA-PEGD-GNPs and
C-BSA in the C-BSA-PEGD-GNPs, the TGA curves of PEGD-GNPs,
BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs were recorded (Fig. 4a–c).
Water desorption between room temperature and ∼110 ◦ C was
considered in these estimations. The amount of PEGD was estimated to be 28.0% from the TGA curve of PEGD-GNPs (Fig. 4a).
The amounts of BSA-PEGD and C-BSA-PEGD were estimated to be
47.4 and 61.7% from the TGA curves of BSA-PEGD-GNPs and CBSA-PEGD-GNPs, respectively (Fig. 4b and c). The amounts of BSA
and C-BSA were estimated to be 19.4 and 33.7% by subtracting
the amount of PEGD from those of PEGD-BSA and C-BSA-PEGD,
respectively. Using the davg of 2.0 nm for the ultrasmall GNPs estimated from the HVEM image and assuming that their density is
the same as that (=7.407 g mL−1 ) [34] of bulk Gd2 O3 , the number of GNPs conjugated to each BSA and C-BSA were estimated
to be 8.8 and 0.2, respectively, which were consistent with HVEM
observations (Fig. 1b and c). Therefore, BSA is a good nanoparticle carrier, but C-BSA is not. This can be explained using the sizes
and masses of BSA and C-BSA. That is, the mass of ultrasmall GNPs
with the davg = 2.0 nm was estimated to be 10–20 kD by calculating
the volume of the ultrasmall GNPs and using the bulk density of
Gd2 O3 [34], which are smaller than those of BSA (mass = 66.5 kD
and size = 14 × 4 × 4 nm). On the other hand, C-BSAs with masses
of 6.67 and 2.01 kD estimated from GPC have sizes of 6.5 × 1.9 × 1.9
3.3. Suggested structures of BSA-PEGD-GNP and
C-BSA-PEGD-GNP
As mentioned before, the conjugation between PEGD-GNP and
BSA (or C-BSA) is an amide bond between –COOH of PEGD-GNP
and –NH2 of BSA (or C-BSA). The BSA consists of 607 amino
acids and has amino acids with a free –NH2 [35,36] that can
be used for the amide bonding to PEGD-GNP. In fact, 60 lysines
with a free –NH2 are in BSA. Therefore, there are plenty of free
–NH2 in BSA which can be conjugated to PEGD-GNPs through the
amide bonding. As described previously, ∼9 PEGD-GNPs were estimated to be conjugated to each BSA whereas ∼0.2 PEGD-GNPs,
to each C-BSA. Based on these results, structures of the BSAPEGD-GNP and C-BSA-PEGD-GNP were schematically drawn in
Scheme 1c.
3.4. Relaxivities and map images
Magnetic properties of gadolinium oxide nanoparticles have
been well characterized [37]. They are paramagnetic but have
an appreciable magnetic moment at room temperature. This is
because Gd(III) has seven unpaired 4f-electrons (8 S7/2 ). Therefore,
appreciable r1 and r2 values are expected from sample solutions,
which were in fact observed in this study. The r1 and r2 values of BSA-PEGD-GNPs were estimated to be 6.0 s−1 mM−1 and
28.0 s−1 mM−1 , respectively, from the slopes in the plot of 1/T1 and
1/T2 , as a function of the Gd concentration (Fig. 5a). In the same
way, the r1 and r2 values of C-BSA-PEGD-GNPs were estimated to be
7.6 s−1 mM−1 and 22.0 s−1 mM−1 , respectively (Fig. 5b). These values are also listed in Table 1 along with those of the other chemicals
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
73
Table 1
Water proton relaxivity (r1 and r2 )a of various chemicals.
Chemical
d or davg b
Nc
r1
r2
Hd
Te
Ref.
Gd-DTPA
BSA-GNP
BSA-PEGD-GNP
C-BSA-PEGD-GNP
–
20–40
2.0
2.0
–
–
8.8
0.21
4.1
6.7
6.0
7.6
–
38.5
28.0
22.0
0.47
4.7
1.5
1.5
38
37
22
22
[5]
[10]
This work
This work
a
b
c
d
e
Unit: s−1 mM−1 .
Particle diameter or average particle diameter (nm).
Number of GNPs conjugated to a BSA or C-BSA.
Applied MR field (T).
Sample solution temperature (◦ C).
for comparison. The r1 and r2 values of BSA-PEGD-GNPs and C-BSAPEGD-GNPs are larger than those [5,6] of molecular Gd-DTPA. These
increased relaxivities were attributed to the high density of Gd(III)
in the NPs. These larger values generally lead to a higher sensitivity
for detecting diseases in the body through contrast enhancements
and can also provide the same quality MR images as those of the Gdchelates at reduced doses. The r2 values are significantly larger than
that of molecular Gd-DTPA, which is why only NPs are eligible as
T2 MRI contrast agents, whereas molecular agents are only suitable
as T1 MRI contrast agents. The r1 and r2 values of BSA-PEGDGNPs and C-BSA-PEGD-GNPs were similar to those [10] of BSA-GNP
(d = 20–40 nm) measured at a higher applied MR field. On the other
hand, considering that the water proton relaxivities increase with
increasing applied MR field, those of the BSA-PEGD-GNPs and CBSA-PEGD-GNPs will be larger than those of BSA-GNP at the same
applied MR field. This is due likely to the particle size effect of
the GNP. Both aqueous solutions of BSA-PEGD-GNPs and C-BSAPEGD-GNPs showed clear dose-dependent contrast enhancements
in their R1 and R2 map images (Fig. 6a and b), suggesting that these
NPs are potential candidates for MRI contrast agents, which were
confirmed in a mouse experiment.
3.5. In vitro cytotoxicity
The in vitro cytotoxicity of the aqueous sample solutions of BSAPEGD-GNPs and C-BSA-PEGD-GNPs were measured using DU145
and NCTC1469 cells with Gd concentrations up to 200 M (Fig. 7a
and b). The results showed that C-BSA-PEGD-GNPs were slightly
less toxic than BSA-PEGD-GNPs. This is probably because many CBSA like polymers encapsulated the PEG-GNPs, as shown in the
HVEM image (Fig. 1c), whereas many PEGD-GNPs were conjugated to each BSA on the surface of BSA, as shown in the HVEM
image (Fig. 1b). Therefore, PEGD-GNPs were better protected in CBSA-PEGD-GNPs than in BSA-PEGD-GNPs. The cell viability of both
samples decreased gradually with increasing Gd concentration. The
cell viability of C-BSA-PEGD-GNPs at 100 M Gd reached more than
70% for both cells, whereas that of BSA-PEGD-GNPs reached ∼60%
for both cells. These levels of cellular toxicity are sufficiently low to
carry out in vivo MRI experiments.
3.6. In vivo 3 T T2 MR images of a mouse
Because BSA could bind many ultrasmall Gd2 O3 NPs, whereas
C-BSA did not, as measured by TGA, an in vivo MRI experiment
120
(a)
DU145
NCTC1469
Cell Viability (%)
100
80
60
40
20
0
0
120
(b)
DU145
NCTC1469
100
Cell Viability (%)
10
100
200
Concentration Gd ( M)
80
60
40
20
0
0
Fig. 6. R1 and R2 map images of aqueous sample solutions of (a) BSA-PEGD-GNPs
and (b) C-BSA-PEGD-GNPs as a function of the Gd concentration.
10
100
200
Concentration Gd ( M)
Fig. 7. In vitro cytotoxicity of aqueous sample solutions of (a) BSA-PEGD-GNPs and
(b) C-BSA-PEGD-GNPs using both DU145 and NCTC1469 cells.
74
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
(2) MR relaxivity measurements revealed that both BSA-PEGDGNPs and C-BSA-PEGD-GNPs had r1 and r2 values larger than
those of molecular Gd-chelates.
(3) The 3 T T2 MR images after injecting an aqueous sample solution of BSA-PEGD-GNPs into the mouse tail vein showed clear
negative contrast enhancements.
(4) Large relaxivities and in vivo T2 MR images prove that BSAPEGD-GNPs and C-BSA-PEGD-GNPs are potential MRI contrast
agents.
These results suggest that biological molecules such as BSA can
be used to conjugate many surface modified ultrasmall NPs which
can be applied to a variety of biomedical areas such as MRI contrast
agents studied in this work.
Acknowledgments
Fig. 8. (a) 3 T T2 MR images of the liver of a mouse before and after injecting an
aqueous sample solution of BSA-PEGD-GNPs into a mouse tail vein and (b) the plot
of signal intensity in T2 MR images as a function of time after injection (0 indicates
“before injection”).
was further carried out using an aqueous sample solution of BSAPEGD-GNPs. Although GNPs are generally used as T1 MRI contrast
agents, the T2 MR images were investigated because the r2 value
was a lot larger than the r1 value, due to the appreciable magnetization of ultrasmall GNPs at room temperature [37]. 250 L
(0.1 mmol Gd/kg) of an aqueous solution of BSA-PEGD-GNPs was
injected into a mouse tail vein and 3 T T2 MR images of the liver
were taken before and after injecting the aqueous sample solution. As shown in Fig. 8a, appreciable negative (or darker) contrast
enhancements were observed in the mouse liver after the injection, which returned to almost the original contrast after 24 h due
likely to the excretion of BSA-PEGD-GNPs. To more clearly see the
time evolution of the contrast change in T2 MR images, the signal
intensity in T2 MR images was plotted as a function of time up to
24 h in Fig. 8b. This plot clearly shows that the negative contrast
enhancement maintained up to 91 min after injection but returned
to almost zero above 91 min due likely to the excretion of BSAPEGD-GNPs. These results clearly indicate that the sample solution
functioned as a T2 MRI contrast agent.
4. Conclusions
In summary, we synthesized PEGD coated ultrasmall Gd2 O3 NPs
(i.e. PEGD-GNPs) which were then conjugated to BSA and C-BSA
through amide bonding (i.e. BSA-PEGD-GNPs and C-BSA-PEGDGNPs). We characterized physical and in vitro MRI properties,
and cytotoxicity of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs, and
obtained in vivo MR images using BSA-PEGD-GNPs.
(1) BSA (Mn = 66.5 kD) could bind many ultrasmall PEGD-GNPs
(the core davg = 2.0 nm), showing that BSA is a good ultrasmall
NP carrier. The TGA showed that ∼9 ultrasmall PEGD-GNPs
could be conjugated to each BSA. However, C-BSAs (Mn < 7 kD)
could not bind many ultrasmall PEGD-GNPs due to its reduced
size and mass. Instead many C-BSAs were conjugated to an
ultrasmall PEGD-GNP like polymers. The TGA showed that ∼5
C-BSAs were conjugated to each ultrasmall PEGD-GNP.
This study was supported by the Basic Science Research Program (Grant no. 2012R1A1B3004241 to KSC, 2011-0015353 to YC,
and 2013R1A1A4A03004511 to GHL) and the Basic Research Laboratory (BRL) program (Grant no. 2013R1A4A1069507) through the
National Research Foundation funded by the Ministry of Education,
Science, and Technology, the R&D program of MKE/KEIT (Grant no.
10040393, development and commercialization of molecular diagnostic technologies for lung cancer through clinical validation), and
the KNU Research Fund (2013). The authors wish to thank the Korea
Basic Science Institute for the use of their HVEM and XRD.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2014.
03.011.
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Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and
Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa
Bovine serum albumin (BSA) and cleaved-BSA conjugated ultrasmall
Gd2 O3 nanoparticles: Synthesis, characterization, and application to
MRI contrast agents
Md. Wasi Ahmad a , Cho Rong Kim a , Jong Su Baeck b , Yongmin Chang b,d,∗ ,
Tae Jeong Kim c,d , Ji Eun Bae d , Kwon Seok Chae d,e , Gang Ho Lee a,d,∗
a
Department of Chemistry, College of Natural Sciences, Kyungpook National University (KNU), Taegu 702-701, South Korea
Department of Molecular Medicine and Medical & Biological Engineering, School of Medicine, KNU, Taegu 702-701, South Korea
c
Department of Applied Chemistry, College of Engineering, KNU, Taegu 702-701, South Korea
d
Department of Nanoscience and Nanotechnology, KNU, Taegu 702-701, South Korea
e
Department of Biology Education, Teacher’s College, KNU, Taegu 702-701, South Korea
b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• BSA and C-BSA conjugated ultrasmall
•
•
•
•
Gd2 O3 nanoparticles were synthesized.
BSA can bind many ultrasmall Gd2 O3
nanoparticles whereas C-BSA cannot.
Large water proton relaxivities were
observed.
High contrast MR images in a mouse
liver after intravenous injection were
observed.
These nanoparticles are potential
MRI contrast agents.
a r t i c l e
i n f o
Article history:
Received 15 November 2013
Received in revised form 23 February 2014
Accepted 1 March 2014
Available online 12 March 2014
Keywords:
BSA
C-BSA
Ultrasmall Gd2 O3 nanoparticle
Nanoparticle carrier
MRI
Contrast agent
a b s t r a c t
Bovine serum albumin (BSA) (Mn = 66.5 kD, size = 14 × 4 × 4 nm) is an attractive biological molecule for
biomedical applications because of its water-solubility and bio-compatibility. It can also bind many
ultrasmall nanoparticles (NPs) as confirmed in this study. We synthesized polyethylene glycol diacid
(PEGD) coated ultrasmall Gd2 O3 nanoparticles (PEGD-GNPs, the core davg = 2.0 nm), which were then conjugated to BSA and cleaved-BSA (C-BSA) (i.e. BSA-PEGD-GNPs and C-BSA-PEGD-GNPs) through amide
bonding. Large relaxivities were observed in both aqueous sample solutions (r1 = 6.0 s−1 mM−1 and
r2 = 28.0 s−1 mM−1 for BSA-PEGD-GNPs and r1 = 7.6 s−1 mM−1 and r2 = 22.0 s−1 mM−1 for C-BSA-PEGDGNPs). Three tesla T2 magnetic resonance imaging (MRI) in a mouse after the injection of an aqueous
sample solution of BSA-PEGD-GNPs into a mouse tail vein revealed significant negative contrast enhancements. Large relaxivities and in vivo MR images prove that BSA-PEGD-GNPs and C-BSA-PEGD-GNPs are
potential MRI contrast agents.
© 2014 Elsevier B.V. All rights reserved.
Abbreviations: BSA, bovine serum albumin; C-BSA, cleaved BSA; PEGD, polyethylene glycol diacid; NP, nanoparticle; GNP, Gd2 O3 NP; BRB, Britton–Robinson buffer; PBS,
phosphate buffer solution; MRI, magnetic resonance imaging; NCT, neutron capture therapy; CT, X-ray computed tomography; DTPA, diethylenetriaminepentaacetic acid; PBS,
phosphate buffer solution; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GPC, gel permeation chromatograph; HVEM, high voltage
electron microscope; XRD, X-ray diffraction; ICPAES, inductively coupled plasma atomic emission spectrometer; FT-IR, Fourier transform-infrared; TGA, thermo-gravimetric
analyzer; DU145, human prostate cancer cell; NCTC1469, normal mouse hepatocyte cell.
∗ Corresponding authors. Tel.: +82 53 950 5340; fax: +82 53 950 6330.
E-mail addresses: ychang@knu.ac.kr (Y. Chang), ghlee@mail.knu.ac.kr (G.H. Lee).
http://dx.doi.org/10.1016/j.colsurfa.2014.03.011
0927-7757/© 2014 Elsevier B.V. All rights reserved.
68
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
1. Introduction
2.2. Synthesis of PEGD coated ultrasmall GNPs (PEGD-GNPs)
Biological molecules have attracted considerable interest for
application to nanomedicine because they are fully biocompatible
and water-soluble. Bovine serum albumin (BSA) is an important
carrier protein in blood plasma for several ions and molecules. It has
a large dimension (14 × 4 × 4 nm) and heavy mass (Mn = ∼66.5 kD).
Thus, it may also carry several ultrasmall nanoparticles (NPs) with
a smaller size and mass than BSA. Biological molecules have several advantages over small molecules and polymers for biomedical
applications. First, the water-solubility of surface-modified NPs
generally increases with increasing mass of the ligands [1,2] and
thus, biological molecules will provide enhanced water-solubility
for NPs. Second, the NPs conjugated to biological molecules can
remain in the blood for a longer duration than free NPs and
Gd-chelates, allowing longer imaging times (so called blood-pool
imaging agents) and a higher likelihood of delivering NPs to the
targeted areas in a body [3–6].
This study makes use of BSA and ultrasmall Gd2 O3 NPs (GNPs)
for magnetic resonance imaging (MRI). Ultrasmall GNPs have
shown longitudinal (r1 ) and transverse (r2 ) water proton relaxivities larger than those of Gd-chelates because of the dense
population of Gd(III) in NPs [7,8]. Therefore, BSA which could bind
several ultrasmall GNPs may be useful for MRI. Here, ultrasmall
GNPs can be also applied to X-ray computed tomography (CT)
as CT contrast agents and neutron capture therapy (NCT) as NCT
agents because Gd has a large X-ray attenuation power (∼2.5 times
stronger than commercial iodine CT contrast agents) [9–12] and a
very large neutron capture cross section (∼254,000 barns) [13–16].
This implies that BSA conjugated ultrasmall GNPs could be also
useful for CT and NCT.
Previous studies on MRI using BSA include BSA[Gd-chelates]n
and BSA-GNPs (d = 20–40 nm) [3–6,10,17]. Enhanced water proton
relaxivities have been observed in both systems after conjugation to BSA. In addition, the in vivo application of BSA[Gd-DTPA]n
(DTPA = diethylenetriaminepentaacetic acid) showed a longer circulation time in the blood than Gd-DTPA, providing longer MR
imaging times in brain tumors and blood vessels in rats [3,4].
This study examined BSA and cleaved BSA (C-BSA) (99.9%), triethylene glycol (99%), polyethylene glycol diacid (PEGD) (Mn = 600),
BSA (Mn = ∼66.5 kD), phosphate buffer solution (PBS) (pH = 7.2),
HCl (>99%), N-hydroxysuccinimide (NHS) (98%), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) (>97%), boric acid
(>98%), phosphoric acid (>97%), and acetic acid (98%) were purchased from Sigma-Aldrich and used as received. Triply distilled
water was used for both washing the products and preparing the
MRI sample solutions.
2.3. Synthesis of C-BSA
To prepare C-BSA, 10 mmol of l-serine and 10 mmol of lhistidine were dissolved in 300 mL of a Britton–Robinson buffer
(BRB) (40 mM phosphate, 40 mM acetate, and 40 mM borate)
(pH = 5–6) at 60 ◦ C and under atmospheric conditions (Scheme 1b)
[19]. Here, a pH of 5–6 of the buffer solution was achieved by adding
NaOH slowly to the original buffer solution. After magnetic stirring
for 30 min, 156 mg of BSA was added to the solution, and the solution was stirred magnetically for 36 h at 60 ◦ C. After the reaction
was complete, the water was evaporated. This cleavage reaction
was repeated 10 times to obtain enough C-BSA. The masses of CBSAs were characterized by gel permeation chromatography (GPC)
and the result is summarized in Supporting Information. Two major
C-BSAs with masses of 6.67 and 2.01 kD were observed from GPC
analysis. The C-BSA was used without further purification because
the water-soluble C-BSA could not be separated from other watersoluble reagents used in the reaction. On the other hand, the water
soluble reagents were later removed after conjugation of C-BSAs to
PEGD-GNPs through amide bonding.
2.4. Synthesis of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs
BSA-PEGD-GNPs and C-BSA-PEGD-GNPs were synthesized
using an EDC/NHS coupling method (Scheme 1c) [20,21]. In this
reaction amide bonds were formed between –COOH of PEGD-GNPs
and –NH2 of BSA (or C-BSA). The reaction was carried out at room
temperature and under atmospheric conditions. Solution pH was
fixed at 6.0 by adding 1 mM HCl to the original PBS with pH of 7.2.
5 mmol of EDC and 5 mmol of NHS were added to 30 mL of PBS
(pH = 6). After magnetic stirring for 30 min, the PEGD-GNPs were
added to the solution and the solution was stirred magnetically
for 2 h. 1.5 g of BSA (or C-BSA) was added to the solution at room
temperature and stirred for an additional 2 h. The product solution
was transferred to a 1 L beaker containing 500 mL of triply distilled
water. The resulting solution was stirred magnetically for 10 min
and stored for a week until BSA-PEGD-GNPs (or C-BSA-PEGD-GNPs)
settled in the bottom of the beaker. The supernatant was decanted
and the remaining sample solution was washed again with triply
distilled water. This procedure was repeated three times. During
this process, any water-soluble impurities such as solvent and any
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
69
Scheme 1. Syntheses of (a) the ultrasmall GNPs and PEGD-GNPs, (b) the C-BSA, and (c) the BSA-PEGD-GNPs and C-BSA-PEGD-GNPs.
other reagents used in synthesis of C-BSA, were removed from the
products. Portions of the samples were evaporated to a powder
form in air and the remaining portions were diluted with triply
distilled water to prepare aqueous sample solutions for the MRI
experiments.
2.5. General characterization
The particle diameters of the ultrasmall GNPs were measured
with a high voltage electron microscope (HVEM) (JEOL JEMARM1300S, 1.2 MeV acceleration voltage). A copper grid (PELCO
No. 160, TED PELLA, INC.) covered with an amorphous carbon
membrane was placed onto a filter paper and a sample solution diluted in triply distilled water was dropped onto the copper
grid using a micropipette (Eppendorf, 2–20 L). The copper grid
was dried in air for one hour to remove the solvent. The crystal structure of ultrasmall GNPs was examined using an X-ray
diffraction (XRD) spectrometer (Philips, X-PERT PRO MRD) with
unfiltered Cu-K␣ radiation of 1.54184 Å, scanning step of 0.033◦ ,
and scan range of 2 = 15–100◦ . The concentration of Gd in the sample solution was determined using an inductively coupled plasma
atomic emission spectrometer (ICPAES) (Thermo Jarrell Ash Co.,
IRIS/AP). The surface coating of the ultrasmall GNPs with PEGD
and conjugation of PEGD-GNPs to BSA (or C-BSA) were investigated
using a Fourier transform-infrared (FT-IR) absorption spectrometer (Mattson Instruments, Inc., Galaxy 7020A). The powder samples
were dried on a hot plate at 50 ◦ C in a hood for one week to
minimize the water content. To record the FT-IR absorption spectra (400–4000 cm−1 ), pellets of the powder samples in KBr were
prepared. The amount of surface coating was estimated with a
thermo-gravimetric analyzer (TGA) (TA Instruments, SDT-Q 600).
The TGA curves of the powder samples were recorded between
room temperature and 700 ◦ C while air flowed. The amounts of
PEGD, BSA, and C-BSA per GNP were estimated by recording the TGA
curves. Water desorption between room temperature and ∼110 ◦ C
was considered in these estimations.
70
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
2.6. Relaxivity and map image measurement
Both the longitudinal (T1 ) and transverse (T2 ) relaxation times
and longitudinal (R1 ) and transverse (R2 ) map images of the
aqueous sample solutions of BSA-PEGD-GNPs and C-BSA-PEGDGNPs were measured using a 1.5 T magnetic resonance imaging
(MRI) instrument (GE 1.5 T Signa Advantage, GE medical system)
equipped with a Knee coil (EXTREM). Aqueous sample solutions at
different Gd concentrations were prepared by diluting the original sample solutions with triply distilled water. Both map images
and relaxation times were measured using these solutions. The longitudinal (r1 ) and transverse (r2 ) water proton relaxivities were
estimated from the slopes in the plots of 1/T1 and 1/T2 versus
the Gd concentration, respectively. The typical parameters used to
measure the relaxation times and map images were as follows:
the external MR field (H) = 1.5 T, the temperature (T) = 22 ◦ C, the
number of acquisition (NEX) = 1, the field of view (FOV) = 16 cm,
the phase FOV = 1, the matrix size = 512 × 512, the slice thickness = 5 mm, the spacing gap = 0, the pixel bandwidth = 61.0547, the
repetition time (TR) = 2009 ms, and the echo time (TE) = 9 ms.
2.7. In vitro cytotoxicity measurement
The in vitro cytotoxicity of the aqueous sample solutions of
BSA-PEGD-GNPs and C-BSA-PEGD-GNPs was measured using both
human prostate cancer (DU145) and normal mouse hepatocyte
(NCTC1469) cells. A CellTiter-Glo Luminescent Cell Viability Assay
(Promega, WI, USA) was used to measure the cytotoxicity. In this
assay, the intracellular ATP was quantified using a luminometer
(Victor 3, Perkin Elmer). The cells were seeded onto a 24-well cell
culture plate and incubated for 24 h (5 × 104 cell density, 500 L
cells per well, 5% CO2 , 37 ◦ C). A series of test sample solutions (0,
10, 100, and 200 M) were prepared by diluting the original sample
solutions with a sterile phosphate buffer saline solution. ∼2 mL of
each test solution was added to the cell culture media. The treated
cell culture media were then incubated for 48 h. The cell viability of
each cell was determined and normalized with respect to that of the
control cell with 0.0 M Gd concentration. The measurements for all
test cells were repeated twice to obtain the average cell viabilities.
2.8. In vivo 3 T T2 MR image measurement
A 3 T MRI instrument (SIEMENS 3.0 T MAGNETOM Trio a Tim)
was used to measure the T2 spin echo (SE) images of a mouse.
The animal experiment in this study was carried out under the
permission and guidance of the KNU animal committee. An ICR
female mouse (ICR—Institute of Cancer Research, USA) with a
weight of ∼100 g was used for the MR image measurements.
The mouse was anesthetized by 1.5% isoflurane in oxygen. The
measurements were made before and after injecting the sample
solution into a mouse tail vein. The injection dose was ∼250 L
(∼0.1 mmol Gd kg−1 ). After the measurement, the mouse was
revived from anesthesia, placed into a cage, and given a free access
to both food and water. During the measurement, each mouse
was maintained at ∼37 ◦ C using a warm water blanket. The typical measurement parameters were as follows: the H = 3 T, the
Fig. 1. HVEM images of (a) and (b) ultrasmall GNPs at two different scales, (c) BSA-PEGD-GNPs, and (d) C-BSA-PEGD-GNPs.
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
71
3. Results and discussion
through hydrogen bonding [23–25]. The estimated cell constants
are a = 6.644 Å, b = 6.841 Å, c = 6.328 Å, and ˇ = 103.976◦ for TGAtreated BSA-PEGD-GNPs and a = 6.643 Å, b = 6.839 Å, c = 6.326 Å, and
ˇ = 104.001◦ for TGA-treated C-BSA-PEGD-GNPs which are consistent with the values reported in PCPDFWIN [26].
3.1. Particle diameter (d) and crystal structure of ultrasmall GNPs
3.2. Surface modification of ultrasmall GNPs
Fig. 1a–d shows HVEM images of the as-prepared ultrasmall GNPs, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs. The particle
diameters of the ultrasmall GNPs ranged from 1 to 3 nm with the
davg of 2.0 nm (Fig. 1a and b). The HVEM image of BSA-PEGD-GNPs
indicated that many ultrasmall PEGD-GNPs were conjugated to a
BSA (Fig. 1c). On the other hand, the HVEM image of C-BSA-PEGDGNPs showed that many C-BSAs were conjugated to each ultrasmall
PEGD-GNP (Fig. 1d). As will be discussed later, this is consistent
with the numbers of PEGD-GNPs conjugated to BSA and C-BSA
estimated from TGA analyses.
The XRD patterns of powder samples of both BSA-PEGD-GNPs
and C-BSA-PEGD-GNPs were measured before and after TGA and
are provided in Fig. 2. The XRD patterns of the as-prepared powder samples were broad, due likely to ultrasmall particle diameters
[22]. On the other hand, the XRD patterns of the TGA-treated powder samples revealed sharp peaks, corresponding to monoclinic
GdPO4 . All peaks after TGA analysis could be assigned to monoclinic
GdPO4 and the peak positions with sufficient intensities are marked
with ‘*’ in Fig. 2 and the Miller index (h k l) assignments of these
peaks are provided in Supporting information. The formation of
GdPO4 after TGA analysis is because the EDC/NHS coupling reaction
was carried out in PBS. That is, the PO4 3− ions were likely attached
to amine groups of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs
The surface coating of ultrasmall GNPs with PEGD followed by
conjugation to BSA or C-BSA was investigated by FT-IR absorption
spectroscopy. As mentioned previously, one group among the two
–COOH groups in each PEGD was conjugated to an ultrasmall GNP
and the other was left free for amide bonding to BSA or C-BSA.
These were confirmed from the two different C=O stretching
vibrations in the FT-IR absorption spectrum of the PEGD-GNPs
(Fig. 3a). The free –COOH was observed at 1730 cm−1 but the
–COOH bonded to GNPs, at 1620 cm−1 . The peaks at 2910 cm−1
(C–H stretch) and 1110 cm−1 (C–O stretch) also confirmed that the
PEGDs were bonded to ultrasmall GNPs. The peak at 3420 cm−1
in the PEGD-GNPs was assigned to the water –OH stretch. The
∼110 cm−1 red shift of the C=O stretch after bonding to GNPs
from that of the free –COOH had been observed in a range of
the metal oxide NPs coated with the ligands with –COOH groups
[22,27–30], supporting this result. The successful amide bond
formation between PEGD-GNPs and BSA (or C-BSA) was confirmed
from the disappearance of a free C=O stretch at 1730 cm−1 in both
BSA-PEGD-GNPs and C-BSA-PEGD-GNPs (Fig. 3b). Instead, the N–H
stretch at 3440 cm−1 (overlapped with water –OH stretch) and the
N–H bend at 1530 cm−1 were observed [31–33]. The reduced intensity in N–H bend in both BSA-PEGD-GNPs and C-BSA-PEGD-GNPs
was, however, observed owing to the amide bonding of amine
groups of BSA and C-BSA with PEGD-GNPs and the hydrogen
bonding of amine groups of BSA and C-BSA with PO4 3− ions.
T = 37 ◦ C, the NEX = 3–4, the FOV = 60 mm, the phase FOV = 30 mm,
the matrix size = 128 × 256, the slice thickness = 1 mm, the spacing
gap = 0.1 mm, the TR = 2690 ms, and the TE = 37 ms.
*
* : GdPO4
(a)
*
*
after TGA
*
*
*
*
*
*
*
* *
*
*
*
*
*
*
*
*
as prepared
20
40
60
Transmittance (%)
Intensity (Arb. Units)
(a)
PEGD-GNP
1730
3420 2910
2
*
1620
1110
3000
2000
1000
-1
Wavenumber (cm )
* : GdPO4
(b)
*
*
*
*
after TGA
*
**
*
*
*
*
*
*
*
*
*
*
*
as prepared
20
40
60
2
Fig. 2. XRD patterns of powder samples of (a) BSA-PEGD-GNPs and (b) C-BSA-PEGDGNPs before (i.e. as-prepared) and after TGA analysis. All peaks after TGA analysis
could be assigned to monoclinic GdPO4 and only the peak positions with sufficient intensities are marked with ‘*’ (Miller index (h k l) assignments are provided
in Supporting information).
Transmittance (%)
Intensity (Arb. Units)
4000
*
(b)
PEGD
BSA
BSA-PEGD-GNP
2910
1620 1530
C-BSA-PEGD-GNP
3440
4000
3000
2000
1000
-1
Wavenumber (cm )
Fig. 3. FT-IR absorption spectra of powder samples of (a) PEGD and PEGD-GNPs and
(b) BSA, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs.
72
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
100
30
Water desorption = 9.8%
(a)
(a)
28.0%
60
20
-1
40
10
-1
0
-1
r1 = 6.0 s mM
20
0
0
0.00
100 200 300 400 500 600 700
o
Temperature ( C)
100
Water desorption = 4.9%
0.25
0.50
0.75
Concentration Gd (mM)
1.00
6
(b)
80
5
47.4%
(b)
-1
-1
r2 = 22.0 s mM
4
-1
)
60
1/T (s
Weight (%)
-1
r2 = 28.0 s mM
-1
1/T (s )
Weight (%)
80
40
20
3
-1
-1
r1 = 7.6 s mM
2
1
0
0
100
(c)
100 200 300 400 500 600 700
o
Temperature ( C)
Weight (%)
61.7%
0.04 0.08 0.12 0.16
Concentration Gd (mM)
0.20
Fig. 5. Plots of 1/T1 and 1/T2 of the aqueous sample solutions of (a) BSA-PEGDGNPs and (b) C-BSA-PEGD-GNPs as a function of the Gd concentration. The slopes
correspond to the r1 and r2 values, respectively.
and 4.4 × 1.2 × 1.2, respectively, assuming a cube root dependence
of the size on the mass. Therefore, C-BSA is not large enough in
mass and size to bind many ultrasmall GNPs, which is similar to
the polymers.
40
20
0
0.00
Water desorption = 7.3%
80
60
0
0
100 200 300 400 500 600 700
o
Temperature ( C)
Fig. 4. TGA curves of powder samples of (a) PEGD-GNPs, (b) BSA-PEGD-GNPs, and
(c) C-BSA-PEGD-GNPs.
To determine the amounts of BSA in the BSA-PEGD-GNPs and
C-BSA in the C-BSA-PEGD-GNPs, the TGA curves of PEGD-GNPs,
BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs were recorded (Fig. 4a–c).
Water desorption between room temperature and ∼110 ◦ C was
considered in these estimations. The amount of PEGD was estimated to be 28.0% from the TGA curve of PEGD-GNPs (Fig. 4a).
The amounts of BSA-PEGD and C-BSA-PEGD were estimated to be
47.4 and 61.7% from the TGA curves of BSA-PEGD-GNPs and CBSA-PEGD-GNPs, respectively (Fig. 4b and c). The amounts of BSA
and C-BSA were estimated to be 19.4 and 33.7% by subtracting
the amount of PEGD from those of PEGD-BSA and C-BSA-PEGD,
respectively. Using the davg of 2.0 nm for the ultrasmall GNPs estimated from the HVEM image and assuming that their density is
the same as that (=7.407 g mL−1 ) [34] of bulk Gd2 O3 , the number of GNPs conjugated to each BSA and C-BSA were estimated
to be 8.8 and 0.2, respectively, which were consistent with HVEM
observations (Fig. 1b and c). Therefore, BSA is a good nanoparticle carrier, but C-BSA is not. This can be explained using the sizes
and masses of BSA and C-BSA. That is, the mass of ultrasmall GNPs
with the davg = 2.0 nm was estimated to be 10–20 kD by calculating
the volume of the ultrasmall GNPs and using the bulk density of
Gd2 O3 [34], which are smaller than those of BSA (mass = 66.5 kD
and size = 14 × 4 × 4 nm). On the other hand, C-BSAs with masses
of 6.67 and 2.01 kD estimated from GPC have sizes of 6.5 × 1.9 × 1.9
3.3. Suggested structures of BSA-PEGD-GNP and
C-BSA-PEGD-GNP
As mentioned before, the conjugation between PEGD-GNP and
BSA (or C-BSA) is an amide bond between –COOH of PEGD-GNP
and –NH2 of BSA (or C-BSA). The BSA consists of 607 amino
acids and has amino acids with a free –NH2 [35,36] that can
be used for the amide bonding to PEGD-GNP. In fact, 60 lysines
with a free –NH2 are in BSA. Therefore, there are plenty of free
–NH2 in BSA which can be conjugated to PEGD-GNPs through the
amide bonding. As described previously, ∼9 PEGD-GNPs were estimated to be conjugated to each BSA whereas ∼0.2 PEGD-GNPs,
to each C-BSA. Based on these results, structures of the BSAPEGD-GNP and C-BSA-PEGD-GNP were schematically drawn in
Scheme 1c.
3.4. Relaxivities and map images
Magnetic properties of gadolinium oxide nanoparticles have
been well characterized [37]. They are paramagnetic but have
an appreciable magnetic moment at room temperature. This is
because Gd(III) has seven unpaired 4f-electrons (8 S7/2 ). Therefore,
appreciable r1 and r2 values are expected from sample solutions,
which were in fact observed in this study. The r1 and r2 values of BSA-PEGD-GNPs were estimated to be 6.0 s−1 mM−1 and
28.0 s−1 mM−1 , respectively, from the slopes in the plot of 1/T1 and
1/T2 , as a function of the Gd concentration (Fig. 5a). In the same
way, the r1 and r2 values of C-BSA-PEGD-GNPs were estimated to be
7.6 s−1 mM−1 and 22.0 s−1 mM−1 , respectively (Fig. 5b). These values are also listed in Table 1 along with those of the other chemicals
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
73
Table 1
Water proton relaxivity (r1 and r2 )a of various chemicals.
Chemical
d or davg b
Nc
r1
r2
Hd
Te
Ref.
Gd-DTPA
BSA-GNP
BSA-PEGD-GNP
C-BSA-PEGD-GNP
–
20–40
2.0
2.0
–
–
8.8
0.21
4.1
6.7
6.0
7.6
–
38.5
28.0
22.0
0.47
4.7
1.5
1.5
38
37
22
22
[5]
[10]
This work
This work
a
b
c
d
e
Unit: s−1 mM−1 .
Particle diameter or average particle diameter (nm).
Number of GNPs conjugated to a BSA or C-BSA.
Applied MR field (T).
Sample solution temperature (◦ C).
for comparison. The r1 and r2 values of BSA-PEGD-GNPs and C-BSAPEGD-GNPs are larger than those [5,6] of molecular Gd-DTPA. These
increased relaxivities were attributed to the high density of Gd(III)
in the NPs. These larger values generally lead to a higher sensitivity
for detecting diseases in the body through contrast enhancements
and can also provide the same quality MR images as those of the Gdchelates at reduced doses. The r2 values are significantly larger than
that of molecular Gd-DTPA, which is why only NPs are eligible as
T2 MRI contrast agents, whereas molecular agents are only suitable
as T1 MRI contrast agents. The r1 and r2 values of BSA-PEGDGNPs and C-BSA-PEGD-GNPs were similar to those [10] of BSA-GNP
(d = 20–40 nm) measured at a higher applied MR field. On the other
hand, considering that the water proton relaxivities increase with
increasing applied MR field, those of the BSA-PEGD-GNPs and CBSA-PEGD-GNPs will be larger than those of BSA-GNP at the same
applied MR field. This is due likely to the particle size effect of
the GNP. Both aqueous solutions of BSA-PEGD-GNPs and C-BSAPEGD-GNPs showed clear dose-dependent contrast enhancements
in their R1 and R2 map images (Fig. 6a and b), suggesting that these
NPs are potential candidates for MRI contrast agents, which were
confirmed in a mouse experiment.
3.5. In vitro cytotoxicity
The in vitro cytotoxicity of the aqueous sample solutions of BSAPEGD-GNPs and C-BSA-PEGD-GNPs were measured using DU145
and NCTC1469 cells with Gd concentrations up to 200 M (Fig. 7a
and b). The results showed that C-BSA-PEGD-GNPs were slightly
less toxic than BSA-PEGD-GNPs. This is probably because many CBSA like polymers encapsulated the PEG-GNPs, as shown in the
HVEM image (Fig. 1c), whereas many PEGD-GNPs were conjugated to each BSA on the surface of BSA, as shown in the HVEM
image (Fig. 1b). Therefore, PEGD-GNPs were better protected in CBSA-PEGD-GNPs than in BSA-PEGD-GNPs. The cell viability of both
samples decreased gradually with increasing Gd concentration. The
cell viability of C-BSA-PEGD-GNPs at 100 M Gd reached more than
70% for both cells, whereas that of BSA-PEGD-GNPs reached ∼60%
for both cells. These levels of cellular toxicity are sufficiently low to
carry out in vivo MRI experiments.
3.6. In vivo 3 T T2 MR images of a mouse
Because BSA could bind many ultrasmall Gd2 O3 NPs, whereas
C-BSA did not, as measured by TGA, an in vivo MRI experiment
120
(a)
DU145
NCTC1469
Cell Viability (%)
100
80
60
40
20
0
0
120
(b)
DU145
NCTC1469
100
Cell Viability (%)
10
100
200
Concentration Gd ( M)
80
60
40
20
0
0
Fig. 6. R1 and R2 map images of aqueous sample solutions of (a) BSA-PEGD-GNPs
and (b) C-BSA-PEGD-GNPs as a function of the Gd concentration.
10
100
200
Concentration Gd ( M)
Fig. 7. In vitro cytotoxicity of aqueous sample solutions of (a) BSA-PEGD-GNPs and
(b) C-BSA-PEGD-GNPs using both DU145 and NCTC1469 cells.
74
Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75
(2) MR relaxivity measurements revealed that both BSA-PEGDGNPs and C-BSA-PEGD-GNPs had r1 and r2 values larger than
those of molecular Gd-chelates.
(3) The 3 T T2 MR images after injecting an aqueous sample solution of BSA-PEGD-GNPs into the mouse tail vein showed clear
negative contrast enhancements.
(4) Large relaxivities and in vivo T2 MR images prove that BSAPEGD-GNPs and C-BSA-PEGD-GNPs are potential MRI contrast
agents.
These results suggest that biological molecules such as BSA can
be used to conjugate many surface modified ultrasmall NPs which
can be applied to a variety of biomedical areas such as MRI contrast
agents studied in this work.
Acknowledgments
Fig. 8. (a) 3 T T2 MR images of the liver of a mouse before and after injecting an
aqueous sample solution of BSA-PEGD-GNPs into a mouse tail vein and (b) the plot
of signal intensity in T2 MR images as a function of time after injection (0 indicates
“before injection”).
was further carried out using an aqueous sample solution of BSAPEGD-GNPs. Although GNPs are generally used as T1 MRI contrast
agents, the T2 MR images were investigated because the r2 value
was a lot larger than the r1 value, due to the appreciable magnetization of ultrasmall GNPs at room temperature [37]. 250 L
(0.1 mmol Gd/kg) of an aqueous solution of BSA-PEGD-GNPs was
injected into a mouse tail vein and 3 T T2 MR images of the liver
were taken before and after injecting the aqueous sample solution. As shown in Fig. 8a, appreciable negative (or darker) contrast
enhancements were observed in the mouse liver after the injection, which returned to almost the original contrast after 24 h due
likely to the excretion of BSA-PEGD-GNPs. To more clearly see the
time evolution of the contrast change in T2 MR images, the signal
intensity in T2 MR images was plotted as a function of time up to
24 h in Fig. 8b. This plot clearly shows that the negative contrast
enhancement maintained up to 91 min after injection but returned
to almost zero above 91 min due likely to the excretion of BSAPEGD-GNPs. These results clearly indicate that the sample solution
functioned as a T2 MRI contrast agent.
4. Conclusions
In summary, we synthesized PEGD coated ultrasmall Gd2 O3 NPs
(i.e. PEGD-GNPs) which were then conjugated to BSA and C-BSA
through amide bonding (i.e. BSA-PEGD-GNPs and C-BSA-PEGDGNPs). We characterized physical and in vitro MRI properties,
and cytotoxicity of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs, and
obtained in vivo MR images using BSA-PEGD-GNPs.
(1) BSA (Mn = 66.5 kD) could bind many ultrasmall PEGD-GNPs
(the core davg = 2.0 nm), showing that BSA is a good ultrasmall
NP carrier. The TGA showed that ∼9 ultrasmall PEGD-GNPs
could be conjugated to each BSA. However, C-BSAs (Mn < 7 kD)
could not bind many ultrasmall PEGD-GNPs due to its reduced
size and mass. Instead many C-BSAs were conjugated to an
ultrasmall PEGD-GNP like polymers. The TGA showed that ∼5
C-BSAs were conjugated to each ultrasmall PEGD-GNP.
This study was supported by the Basic Science Research Program (Grant no. 2012R1A1B3004241 to KSC, 2011-0015353 to YC,
and 2013R1A1A4A03004511 to GHL) and the Basic Research Laboratory (BRL) program (Grant no. 2013R1A4A1069507) through the
National Research Foundation funded by the Ministry of Education,
Science, and Technology, the R&D program of MKE/KEIT (Grant no.
10040393, development and commercialization of molecular diagnostic technologies for lung cancer through clinical validation), and
the KNU Research Fund (2013). The authors wish to thank the Korea
Basic Science Institute for the use of their HVEM and XRD.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2014.
03.011.
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