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Biomaterials 35 (2014) 5381e5392

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Paramagnetic hollow silica nanospheres for in vivo targeted


ultrasound and magnetic resonance imaging
Lu An a, He Hu a, *, Jing Du b, Jie Wei a, Li Wang a, Hong Yang a, Dongmei Wu c, Haili Shi a,
Fenghua Li b, Shiping Yang a, *
a
The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University,
Shanghai 200234, PR China
b
Department of Ultrasound, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, PR China
c
Shanghai Key Laboratory of Magnetic Resonance, Department of Physics, East China Normal University, Shanghai 200062, PR China

a r t i c l e i n f o a b s t r a c t

Article history: A series of hollow silica nanospheres (HSNSs) with sizes ranging from 100 to 400 nm were synthesized
Received 20 February 2014 and used for primary ultrasound imaging (US) efficiency assessment. The 400 nm HSNSs were chosen as
Accepted 13 March 2014 platform for conjugation with Gd-DTPA and cyclo-arginine-glycine-aspartic acid c(RGD) peptide to
Available online 3 April 2014
construct US and magnetic resonance imaging (MRI) dual-modal contrast agents (CAs): [HSNSs@(DTPA-
Gd)-RGD]. The obtained CAs displayed good physiological stability, low cytotoxicity and negligible he-
Keywords:
molytic activity in vitro. Furthermore, the passive accumulation and active-targeting of the HSNSs in the
Hollow silica nanospheres
tumor site of mice was demonstrated by US and MR imaging, respectively. The qualitative and quanti-
Ultrasound imaging
Magnetic resonance imaging
tative biodistribution of the HSNSs showed that they mainly accumulated in the tissues of liver, lung,
Biodistribution tumor after intravenous administration and then be excreted from feces. In addition, histological, he-
Toxicity matological, blood and biochemical analysis were used to further study toxicity of the HSNSs, and all
results indicated that there were no covert toxicity of HSNSs in mice after long exposure times. Findings
from this study indicated that the silica-based paramagnetic HSNSs can be used as a platform for long-
term targeted imaging and therapy studies safely in vivo.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction [5e7]. In addition, the use of MRI/optical dual-modality imaging


provides both good spatial resolution and planar resolution [8e11].
Recently, a great deal of interest has been focused on the ac- Among the current imaging techniques, ultrasound (US) is a
curate detection of diseases at an early stage by developing real-time imaging mode. It is noninvasive, inexpensive and
multimodal imaging techniques because each single-modality im- portable, and widely used clinically, but it still suffer poor tissue
aging techniques have their own disadvantages and limitations [1e discrimination ability [12,13]. MRI is one of the most powerful and
3]. For example, magnetic resonance imaging (MRI) provides noninvasive diagnostic tools. It can provide images with excellent
excellent anatomical information, but suffers from limited sensi- anatomical detail based on the interaction of protons with the
tivity. Positron emission tomography (PET) has high sensitivity but surrounding molecules of tissues, while cannot provide real-time
low spatial resolution. The combinations of various imaging tech- images and usually need a relatively long imaging time. [14] The
niques could provide more complementary, synergetic and accu- current MRI CAs are generally in the form of T1-positive agents of
rate information about the physical, anatomical structure and the paramagnetic species and T2-negative agents of super-
physiological function for diagnosis and research [4]. Therefore, it is paramagnetic particles. Most presently available T1 CAs are Gd and
desirable to combine different imaging modalities into one single Mn-based complexes or nanoparticles. Thermodynamically stable,
nanoparticle. Recently, some researchers have combined MRI with water-soluble, and high paramagnetic Gd (III) complexes, such as
PET, and achieved excellent spatial resolution and high sensitivity Gd-DOTA and Gd-DTPA, have demonstrated high relaxivity which
has been commercialized as clinical T1-CAs [15]. However, the
short blood circulation time and lack of target ligands is their fatal
* Corresponding authors. Tel./fax: þ86 21 64322343.
E-mail addresses: huheandy@shnu.edu.cn (H. Hu), shipingy@shnu.edu.cn
weakness. Anchoring Gd (III) complexes onto the surface of
(S. Yang). nanoparticles can avoid this defect [16]. As well known, in

http://dx.doi.org/10.1016/j.biomaterials.2014.03.030
0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
5382 L. An et al. / Biomaterials 35 (2014) 5381e5392

practical diagnosis process, US is commonly exploited for a first 2. Experiment section


tumor localization, if necessary, MRI will be done for a second- 2.1. Synthesis of polystyrene (PS) spheres with different sizes
level confirmation. Furthermore, US could be again used as a The PS were prepared using emulsifier-free emulsion polymerization method
real-time guidance during surgical resection. All these only [40,41]. PS-100, PS-200, and PS-400 refer to the 100, 200, and 400 nm PS, respec-
needed a single injection of CAs [17]. Therefore, the combination tively. PS-100 was synthesized in ethanol using AIBA as the initiator and AETAC as
of an MR and US imaging contrast agent within one nanoparticle the stabilizer, while PS-200 and PS-400 using PVP as the stabilizer. Taking PS-400 as
an example, 13.0 g St, 1.0 g PVP, and 90.0 g H2O were added to a 250 mL three-neck
platform would take advantage of both MRI, with its three-
flask. The solution was stirred and deoxygenated by bubbling nitrogen for 20 min,
dimensional spatial resolution, and US imaging, with its real- and then slowly heated to 70  C. Then, 10 mL of an aqueous solution containing
time monitoring [18e24]. Currently, most research work focused 0.55 g AIBA was injected to initiate the polymerization. The emulsion was then
on superparamagnetic nanoparticles-loaded polymer or lipo- polymerized for 24 h under nitrogen to form the latex. The obtained 400 nm PS was
dialyzed in ethanol using a cellulose membrane (MwCO ¼ 3500), and the solid
somes microcapusles for US/MRI [24e26]. However, the relatively
content of the PS suspension could be tailored through the addition of ethanol.
low strength of the organic shells, as well as the large micrometer
size and polydispersed size distribution, have become serious 2.2. Synthesis of amino-functionalized hollow silica nanospheres (HSNSs-NH2)
obstacles to possible clinical applications [27,28]. In our previous
Firstly, 5.0 g PS suspension (containing w0.50 g PS) was added to 40 mL ethanol
work, we have successfully demonstrated hollow silica micro- under gently stirring, and the mixture was slowly heated to 50  C. Then 0.5 g TEOS
spheres as a ultrasound CAs for in vivo imaging [27]. Our work was added dropwise to the suspension at a rate of 1 mL/h using a syringe pump,
opened a promising way of silica-based inorganic ultrasound CAs. followed by addition of 5 mL ammonium hydroxide solution (28%) quickly. After the
Afterwards, Shi’s group further developed the hollow silica mixture were hydrolyzed for 30 min, 0.05 g APS was added. The reaction continued
for an additional 24 h and the obtained coreeshell spheres were separated from the
nanospheres for high-intensity focused ultrasound (HIFU) appli- reaction medium by centrifuging, washed several times with deionized water and
cations [28e31]. Most recently, for example, they reported that ethanol. Finally, the product was freeze dried for 24 h [41], and then, the PS tem-
Mn-loaded hollow mesoporous silica with post-infused liquid plates of the PS@SiO2-NH2 coreeshell nanoparticles were removed by hot THF.
perfluorohexane which exhibited great potential for T1-weighted
MRI-guided HIFU cancer surgery [31]. 2.3. Functionalize HSNSs-NH2 with Gd-DTPA and RGD ligand: [HSNSs@(DTPA-Gd)-
RGD]
Compared to the commercial organic microbubbles or lipo-
somes, the silica-based agents present a significantly advantages, For further functionalization of the HSNSs-NH2 with diethylenetriaminepenta-
acetic acid (DTPA) moieties, 15 mg DTPA was dispersed in 20 mL anhydrous DMSO
for instants, easily controlled particle size and modified surface, under stirring, and then EDC and NHS (molar ratio, DTPA: EDC: NHS ¼ 1:1:2.5) were
homogeneous size distribution, robust shell, efficient ultrasound added to the solution. The mixture was stirred at room temperature for 4 h. 20 mg
response, making them very promising for further clinical appli- HSNSs-NH2 (1.12  104 mol g1 of eNH2) in 20 mL DMSO was added to the suf-
cations. However, there still remain in vivo biocompatibility issue of ficiently activated DTPA solution, and stirring overnight at room temperature. The
HSNSs@(DTPA)-NH2 NPs were purified by repeating the centrifugation and washing
this inorganic HSNSs with respect to the understanding of how
steps: 2 times with DMSO, 2 times with deionized water, followed by freeze dried for
these nanospheres interact with biological systems and the envi- 24 h [42].
ronment. To date, most of reports only concerning the cytotoxicity The prepared HSNSs@(DTPA)-NH2 NPs (20 mg) were dispersed in 15 mL HEPES
of solid or mesoporous silica with different size or shapes [32e34]. (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution (pH ¼ 8.0,
However, the low cytotoxicity don’t guarantee on the desired high 0.1 M). 100 mg Gd(NO3)3∙5H2O was added into the particle suspension while stirring,
and then the solution was deoxygenated by bubbling nitrogen and slowly heated to
biocompatibility in vivo. There are only a few studies investigated 40  C. After 12 h, the Gd(III)- loaded HSNSs@(DTPA-Gd)-NH2 were centrifuged and
the dose toxicity of HSNSs in mice. [35] It is worth noting that the re-dispersed in water for three times and then freeze dried.
size of the HSNSs used in the study is less than 200 nm, and they To modify the targeted unit c(RGD) peptide to the remaining surface amine
neither address the in vivo imaging function of HSNSs nor examine groups of the HSNSs@(DTPA-Gd)-NH2, 5 mg 6-maleimidohexanoic acid n-hydrox-
ysuccinimide ester was added to an HEPES buffer solution (0.05 M, 6 mL, pH ¼ 8.0)
the in vivo sequestration and excretion of particles. Therefore, it’s
containing the HSNSs@(DTPA-Gd)-NH2 at room temperature. After stirring for 6 h,
very important to clarify the physiological stability, toxicity and the particles were centrifuged and washed twice with water. The precipitate was
biodistribution of HSNSs with slightly large size of 400 nm using then re-dispersed into an HEPES buffer solution (0.05 M, 6 mL, pH ¼ 7.3) containing
animal models, the preferred system for toxicological evaluation of 10 mg thiolated c(RGD) peptide, while gently stirring at room temperature for 8 h
a multifunctional CAs. under a flow of nitrogen. HSNSs@(DTPA-Gd)-RGD were centrifuged and washed
with water at least three times and then freeze dried for 24 h in vacuum [43].
A major obstacle in cancer diagnosis and therapy is target
discern the specific disease sites in the body. One of the strategies to 2.4. Hemolysis assay
address this problem is modified the surface of nanoparticles with
Human blood stabilized with EDTA was kindly provided by Shanghai Blood
targeting ligands, such as the arginine-glycine-aspartic acid (RGD) Center (obtained from volunteers). The serum was removed from the blood by
has been reported as an effective anb3 integrin acceptor which centrifugation, and the human red blood cells (HRBCs) were then washed five times
expressed during angiogenesis and widely used for nanoparticle with sterile isotonic PBS solution. Then, the cells were diluted to 10 times with PBS.
surface conjugation [36e39]. The diluted HRBCs suspension (0.3 mL) was then mixed with: a) 1.2 mL PBS as a
negative control; b) 1.2 mL deionized water as a positive control; c) 1.2 mL
In this work, a series of HSNSs with sizes ranging from 100 to
HSNSs@(DTPA-Gd)-RGD suspensions at concentrations of 20, 50, 100, 200, 400, 600,
400 nm were synthesized by the hard-template protocol (Scheme 800 mg/mL. The mixtures were then vortexed and then left to stand for 2 h at room
1). The US imaging efficiency of these nanospheres were system- temperature. Afterwards, the samples were centrifuged and diluted to four times.
atically investigated in order to choose a favorable particle size for The absorbance of the supernatants at 541 nm was measured on a Beckman Coulter
further in vivo applications. Then, the 400 nm HSNSs were DU 730 Nucleic Acid/Protein Analyzer. The hemolysis percentages of the samples
were calculated by dividing the difference in absorbance between the sample and
modified with Gd-DTPA and c(RGD) peptide which offered the the negative control by the difference in absorbance between positive and negative
particle paramagnetic and target capability, respectively. More- controls, and multiplying the resulting ratio by 100% [44,45].
over, the tumor-targeting ability of the HSNSs@(DTPA-Gd)-RGD
was also further evaluated by US and MR imaging using tumor- 2.5. MR imaging in solution and relaxivity measurements
bearing mice model. Finally, the in vivo biodistribution of the MR imaging in solution and relaxivity measurements were performed with a
HSNSs was performed to assess their uptake by tumor/tissues and 0.5 T systems (Shanghai Niumag Corporation ration NM120-Analyst).
their clearance from the mice body. The fluctuation in body weight HSNSs@(DTPA-Gd)-RGD were dispersed in water at various Gd concentrations. T1-
weighted MR images were acquired with a conventional spin-echo sequence
of mice, histological assessment, hematological and serum (TR ¼ 2000 ms, TE ¼ 100 ms, the slice thickness ¼ 0.6 mm, the number of
biochemistry assays were also conducted to address the biocom- acquisition ¼ 1). T1 was acquired using inversion recovery sequence. T2 was acquired
patibility issues. using hard pulse CPMG sequence. T1 and T2 measurements were performed using a
L. An et al. / Biomaterials 35 (2014) 5381e5392 5383

Scheme 1. Schematic illustration the formation of HSNSs@(DTPA-Gd)-RGD.

nonlinear fit to changes in the mean signal intensity within each well as a function of was as follow: TR ¼ 500 ms, TE ¼ 12 ms, matrix ¼ 320  320, FOV ¼ 60  60, flip
TR or TE using the provided quantification software. Relaxivity values of r1 and r2 angle ¼ 90 , slice thickness ¼ 1.5 mm.
were determined through the curve fitting of 1/T1 and 1/T2 relaxation time (s1)
versus the Gd concentration (mM).
2.7. US Imaging in vitro and in vivo
In vitro ultrasound imaging was measured in the static state. The Eppendorf tube
2.6. MR imaging in vitro and in vivo
(2 mL) was filled with the physiological saline solution of HSNSs with different
PC3 cells (5  106) were incubated with HSNSs@(DTPA-Gd)-RGD or concentrations (0e1 mg/mL), and then the tube was immerged in a pure water tank.
HSNSs@(DTPA-Gd)-PEG at different concentrations (0, 1, 10, 50, 75, 100 mg/mL in The transducer was coated with ultrasound gel to avoid air background. The ultra-
RPMI 1640) for 6 h in 37  C and 5% CO2. For comparison, HeLa cells (5  106) with low sound imaging was achieved by using a LA435 linear-array transducer under the
anb3 integrin expression were incubated with HSNSs@(DTPA-Gd)-RGD under similar following parameters: the frequency ¼ 6.0 MHz, the medical index (MI) ¼ 0.06. The
conditions. After incubation, the cells were washed with PBS buffer three times, then solid PS@SiO2 (2 mg/mL) were used as the control experiment. All images were
harvested by treatment with a trypsin-EDTA solution (0.25%), and re-suspended in recorded as digital files for subsequent playback and analysis [20,27].
3 mL PBS buffer with a cell density of 1  106 cells/mL to test MR imaging. The spin- In vivo US images were scanned by small animal ultrasound imaging system
echo sequence of T1-weighted MR images was the same as in solution. The amount (Prospect, S-Sharp) before and after injection of HSNSs@(DTPA-Gd)-RGD (5  1010
of Gd uptake in the cells was determined using a high-resolution sector field ICP- capsules mL1) immediately with following parameters: Dynamic range ¼ 50 dB,
AES. Data were acquired at medium resolution (4000) using rhodium (5 ppb) as Gain ¼ 0 dB, Freq ¼ 40 MHz, Depth ¼ 4.1 mm, Power ¼ 35.3%, Fps ¼ 10 Hz. Animals
an internal standard. Digestion of the cells was performed in concentrated nitric acid were anesthetized and injected as described for MR imaging and kept on a heated
at 90  C for 6 h, and the samples were 10 times diluted with H2O before stage to maintain their body temperature. US gel was used as a coupling agent on the
measurements. tumor of the mice. All images were performed by the imaging system as green
To evaluate the targeted MR enhancement of HSNSs@(DTPA-Gd)-RGD, in vivo overlays on the contrast mode.
MR images were performed in the tumor mice model using a 3.0 T systems (3 T
Siemens Magnetom Trio) equipped with a 8 array Loop coil (Siemens Medical Sys-
2.8. Biodistribution
tems, Shanghai Key Lab of MR, Shanghai, China). Mice were anesthetized using
isoflurane (3% loading dose, 1% maintenance dose), and were scanned before and at For biodistribution studies, the mice were anaesthetized with a 10% tri-
different time points (1 h, 4 h) after injection of HSNSs@(DTPA-Gd)-RGD (1 mg/mL) chloroacetaldehyde hydrate (5 mL g1 of animal weight) by intraperitoneal injection,
with a dose of 16 mg/kg body weight through tail vein. The T1-weighted sequence and injection amount of HSNSs was as same as for MR imaging. The mice were
5384 L. An et al. / Biomaterials 35 (2014) 5381e5392

euthanized at 1, 4, and 8 h post-injection and the major organs were collected and
weighted. Digestion of the tissues was performed in digestion solution containing
mixed acids (nitric acid: perchloric acid, V: V ¼ 4: 1) for 12 h, then heated at 90  C for
6 h. Samples were diluted by 10 times in deionized water before measurements. Gd
content in organs was determined by high-resolution sector field ICP-AES. Values
are presented as mean standard deviation for three mice per group.

2.9. In vivo toxicity studies


The four-week-old male athymic nude mice of test group were anaesthetized
and injected HSNSs@(DTPA-Gd)-RGD as described for biodistribution. The nude
mice with no injection were selected as the control group. The body weights of the
mice were recorded for 4 weeks. Blood samples were collected from orbital sinus of
the mice quickly after 1, 4, 8, 24 h and 10 days post-injection and from mice received
no injection for hematology studies. Three hepatic indicators (alanine aminotrans-
ferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST)); three
indicators for kidney functions, (blood urea nitrogen (BUN), blood urea nitrogen (A/
G) rations, creatinine (CREA)); and the complete blood analysis (white blood cells,
red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpus-
cular hemoglobin, mean corpuscular hemoglobin concentration, platelets) were
measured. Values are presented as mean standard deviation for three mice per
group. Upon completion of the blood collection, the mice were sacrificed and tissues Fig. 2. In vitro ultrasound images of HSNSs with different sizes at different
(heart, liver, spleen, lung, and kidney) were excised, fixed in 4% formalin and con- concentration.
ducted with paraffin embedded sections for H&E staining to histopathology analysis.
The slices were observed by Eclipse E800 microscope (Nikon, Japan).

images of the hollow structure show high contrast between the


3. Results and discussion cores and the shells, which confirmed the PS core have been
completely removed, and the homogeneous silica layer still kept
3.1. Synthesis and characterization of hollow silica nanospheres intact.
(HSNSs)
3.2. In vitro ultrasound imaging of HSNSs
The morphology and size of polystyrene spheres (PS), PS@SiO2-
NH2, and HSNSs were observed by FESEM and TEM, as shown in We first need to know which particle size of the as-prepared
Fig. 1. The PS template shows a uniform particle size distribution HSNSs is most favor respond to US radiation. Therefore, the US
with 100 nm (a1), 200 nm (a2), and 400 nm (a3). After coating the images of these three size HSNSs with different particle concen-
PS with a thin layer (w25 nm) of silica, the core/shell structure trations were investigated using Mylab 90 scanner systems. As
could be clearly observed by TEM with slight increments in diam- shown in Fig. 2, US images of the same particle size HSNSs were
eter of 150 nm (b1), 250 nm (b2), and 450 nm (b3). To obtain hollow brighten with increasing of concentration. We also noticed that the
silica nanospheres, the core/shell nanostructure was treated in hot HSNSs displayed a particle size-dependent image enhancement
THF to dissolve the PS core. As shown in Fig. 1c1ec3, the TEM when at a same concentration. As well known, the core dimensions

Fig. 1. Micrographs of nanospheres with different diameters: (a1ea3) FESEM images of PS spheres, (b1eb3) TEM images of PS@SiO2 core/shell nanospheres, (c1ec3) TEM images of
HSNSs.
L. An et al. / Biomaterials 35 (2014) 5381e5392 5385

Fig. 3. (a) T1-weighted MR images of HSNSs@(DTPA-Gd)-RGD (the color changing from black to white indicates the gradual increase of MR signal intensity). (b) A plot of relaxation
rate against Gd concentration of HSNSs@(DTPA-Gd)-RGD.

of HSNSs are very important for US imaging applications because nanometer on the surface of HSNSs@(DTPA-Gd)-RGD (see
the effectiveness of ultrasound CAs is sensitive to the size of the Supporting Information for the calculation method). Conclusive, the
hollow core [25]. Generally, UCAs with a small core size generate a final HSNSs@(DTPA-Gd)-RGD had been successfully prepared.
poor acoustic signal; a larger core size is preferred for a strong
signal [27]. Therefore, it’s reasonable that the strongest US imaging
signal is observed from the 400 nm HSNSs. On the other hand, it has
been reported that the majority of tumors have a pore cutoff size
between 380 and 780 nm. Nanosized CAs in diameter less than
700 nm is able to penetrate blood vessel of tumor and enter cancer
cells [46,47]. Based on these two facts, the 400 nm HSNSs are a good
candidate as US CAs for further in vivo studies.

3.3. Functionalization and relaxivity property of HSNSs@(DTPA-


Gd)-RGD

The 400 nm HSNSs were then further conjugated with Gd-DTPA


and the c(RGD) peptide for active-targeting T1-weighted MRI. The
process of functionalization of HSNSs@(DTPA-Gd)-RGD were sys-
tematically investigated by following characterizations. (1) First, the
FTIR spectra of HSNSs-NH2, HSNSs@(DTPA)-NH2, HSNSs@(Gd-
DTPA)-NH2 and HSNSs@(Gd-DTPA)-RGD shown in Fig. S1 indicated
the surface conjugation of each steps are successful. (2) Second, the
presence of element Gd in the HSNSs@(DTPA-Gd)-NH2 was also
illustrated by the appearance of the Gd photoelectron peaks at a
binding energy of 143 eV for Gd4d in XPS and in EDAX (Fig. S2).
Study of the high-resolution Gd4d5/2 and Gd4d3/2 spectra gave the
well-defined Si2s and Gd4d3/2-Gd4d5/2 components leading to a
surface quantification of 11.58 atomic % of Gd (Table S1), that further
confirmed the successful coating [42]. (3) Then, the surface potential
of the HSNSs in each step were monitored (Fig. S3). Covered with the
negatively charged DTPA ligand, the potential varied from þ42.8 mV
of HSNSs-NH2 to 11.4 mV of HSNSs@(DTPA)-NH2. After complex-
ation with Gd3þ, the surface potential converted to þ34.7 mV, due to
the coordination of Gd3þ with the carboxyl groups of the DTPA
ligand, and resulting in the neutralization of the negative surface
charge [48]. After final labeled with RGD peptide, the surface po-
tential of HSNSs@(DTPA-Gd)-RGD was decreased to near neutral
(þ5.42 mV). (4) Finally, the amount of amine groups on the HSNSs
were quantitative measured which varied from 1.12  104 (HSNSs-
NH2) to 6.38  105 mol g1 (HSNSs@(DTPA-Gd)-NH2), with 43.03%
free amines available on the surface of the particles for further
conjugation with an excess of RGD peptides. Furthermore, since the Fig. 4. (a) The UV/Vis absorption spectra of HSNSs@(DTPA-Gd)-RGD to detect the
presence of hemoglobin at 541 nm (H2O and PBS were used as positive and negative
mean external and internal diameter of HSNSs@(DTPA-Gd)-NH2 are control, respectively). The inset is magnified scale. (b) The hemolysis percentage of
450 and 400 nm, respectively, and the density of SiO2 is 2.32 g cm3, HRBCs. Inset: the photographs of the centrifugal mixtures to observe the presence of
it can be estimated that there are w2 RGD molecules per square hemoglobin in the supernatant.
5386 L. An et al. / Biomaterials 35 (2014) 5381e5392

Fig. 5. Cell viability analysis of human prostate cancer cell line (PC3), human cervical carcinoma cell line (HeLa) and human normal hepatocyte cell line (L02) incubated with
HSNSs@(Gd-DTPA)-RGD at different concentration for 12 and 24 h.

The paramagnetic property of HSNSs@(DTPA-Gd)-RGD was report [27]. As shown in Fig. 4, there was no obvious hemolysis
investigated by measuring the longitudinal relaxation time (T1) of effect from quantitative analysis by measuring the absorbance of
water protons as a function of Gd3þ in an aqueous solution using a the supernatant at 541 nm (hemoglobin). The hemolysis percent-
0.5 T MRI scanner. The HSNSs@(DTPA-Gd)-RGD exhibited an age was only w2.88% even at a high concentration (800 mg mL1) of
obvious signal enhancement on the T1-weighted images in a HSNSs@(DTPA-Gd)-RGD. The corresponding digital photograph of
concentration-dependent manner (Fig. 3a). As the concentration each sample is shown in the inset of Fig. 4b. During the hemolysis
increased, the T1-weighted MRI showed brighter images. The assay experiments, the resulting solution turned visually red (in the
relaxivity (r1) was calculated from the linear fitting of the measured web version) due to hemoglobin released into the solution by he-
1/T1 data versus Gd3þ concentration, which showed a good linear molysis; a denser red color of the corresponding solution means
relationship (Fig. 3b). Based on the slope, the r1 value was estimated higher hemolytic activity. No visible hemolysis effect could be
to be 11.52 mM1 s1, which is higher than that of clinically available observed visually over a broad concentration range of 0e
gadolinium-based agents (Magnevist) under the same conditions 800 mg mL1. The negligible hemolytic activity of HSNSs@(DTPA-
(4.16 mM1 s1). It was reported previously that the r1 values of Gd-
based contrast agents were strengthened when bound to large
molecules, such as proteins and polymers, due to the limited mo-
lecular motion of Gd3þ ions [49]. Hence, it was conceivable that the
enhancement in the r1 value of HSNSs@(DTPA-Gd)-RGD resulted
from the reduced mobility of Gd3þ ions tightly incorporated into
nanospheres, which influences the paramagnetic behavior of the
Gd3þ ions [50,51]. These results indicate that HSNSs@(DTPA-Gd)-
RGD shows a positive contrast effect and could be used as a po-
tential T1-weighted MRI.

3.4. Stability, hemolysis and cytotoxicity of HSNSs@(DTPA-Gd)-RGD

To assess the stability of HSNSs@(DTPA-Gd)-RGD in physiolog-


ical condition for use as US and MRI contrast agent in vivo, the
hydrodynamic sizes of HSNSs@(DTPA-Gd)-RGD incubated in PBS
(pH ¼ 7.4, 0.1 M) and RPMI containing 10% FBS as simulated in vivo
plasma for different time have been analyzed by dynamic light
scattering (DLS). The size of HSNSs@(DTPA-Gd)-RGD did not
change significantly both in PBS buffer solution and RPMI medium
for one week, revealing that HSNSs@(DTPA-Gd)-RGD have excellent
stability under physiological conditions (Fig. S4a). Additionally,
release measurement of Gd3þ ions from HSNSs@(DTPA-Gd)-RGD in
PBS (pH ¼ 5.3 and 7.4, respectively) and RPMI containing 10% FBS at
37  C was carried out. There nearly no free Gd3þ ions were released
from HSNSs@(DTPA-Gd)-RGD in aqueous medium after one week,
as measured by ICP-AES, which further confirmed that
HSNSs@(DTPA-Gd)-RGD is stable enough for in vivo application
(Fig. S4b).
Blood biocompatibility and cytotoxicity are important factors of
Fig. 6. (a) T1-weighted MR images in PC3 and HeLa cells (RGDþ/PC3: PC3 cells incu-
HSNSs@(DTPA-Gd)-RGD used as a contrast agent for in vivo appli- bated with HSNSs@(DTPA-Gd)-RGD, RGDþ/HeLa: HeLa cells incubated with
cations. The blood compatibility of HSNSs@(DTPA-Gd)-RGD was HSNSs@(DTPA-Gd)-RGD, RGD/PC3: PC3 cells incubated with HSNSs@(DTPA-Gd)-
evaluated by the standard hemolysis assay, as in our previous PEG). (b) The Gd uptake content in cells.
L. An et al. / Biomaterials 35 (2014) 5381e5392 5387

Fig. 7. (a1ec1) US images of tumors pre-injection, the positions of tumors were marked by red polygons. Region 1, 2 and 3 are tumors for the intensity measurement, respectively.
(a2ec2) US images of tumors after injected into PC3 and HeLa tumor-bearing mice. (a3ec3) Typical gray scale intensity recording for the inflow spheres in corresponding region.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Gd)-RGD confirmed the excellent biocompatibility of the spheres experiments were designed. The first two groups used
with blood cells, which is favorable for intravenous administration. HSNSs@(DTPA-Gd)-RGD (RGDþ) incubated with the human pros-
The cytotoxicity of the HSNSs@(DTPA-Gd)-RGD was assessed tate cancer PC3 cells with over-expression of anb3 integrins as the
with three cell lines (PC3, HeLa, and L02 cells) using methyl thia- target group, and human cervical carcinoma HeLa cells with low
zolyl tetrazolium (MTT) assay (Fig. 5). The viability of the untreated anb3 integrins expression as the cell control group, respectively. The
cells was used as the control. No significant negative effects on the HSNSs@(DTPA-Gd)-RGD presented a significant positive contrast
viability of the cells were detected after the cells were incubated enhancement on the T1-weighted images in a concentration-
with various concentrations of HSNSs@(DTPA-Gd)-RGD for 12 h dependent manner, as well as in solution. The brightest images
(Fig. 5a). The cell viability still remained above 80% even at the were of the PC3 cells (Fig. 6a, RGDþ/PC3). This is attributed to the
concentration of 200 mg mL1, and then was slightly reduced but over-expression of anb3 integrins on the cell surface, which gives
still above 70% after incubated over 24 h (Fig. 5b). These results rise to the enhanced cellular uptake of the HSNSs@(DTPA-Gd)-RGD,
indicated that HSNSs@(DTPA-Gd)-RGD have low cytotoxicity at the as clearly indicated by the quantitative ICP-AES analysis results
given concentration range, suggesting their potential (Fig. 6b). In contrast, coinciding with the quantitative analysis, the
bioapplications. T1-weighted images of HeLa cells (RGDþ/HeLa) showed poorer
enhancement due to low expression of anb3 integrins on the cell
3.5. Targeted in vitro MR imaging of HSNSs@(DTPA-Gd)-RGD surface. In the last group, the HSNSs@(DTPA-Gd)-PEG which lack of
RGD target ligands (RGD) were used as material control to incu-
To evaluate the capability of the HSNSs@(DTPA-Gd)-RGD as bated with PC3 cells (RGD/PC3). The MR images showed the
noval integrin anb3 targeted contrast agent, three group weakest signal, which further proved that the cellular binding and
5388 L. An et al. / Biomaterials 35 (2014) 5381e5392

uptake of the HSNSs@(DTPA-Gd)-RGD are mediated through the


specific interaction between the RGD ligand and anb3 integrins on
the cell surface. The above results indicate that cells specifically
uptake HSNSs@(DTPA-Gd)-RGD facilitates their use in MRI appli-
cations in biological systems.

3.6. Targeted US and MRI in vivo

Based on the promising US and MRI results in vitro, further


application of HSNSs@(DTPA-Gd)-RGD for targeted dual-modality
US and MR imaging in vivo were investigated with tumor-bearing
mice. As US is a real-time imaging modality and it is usually used
for pre-scanning of disease, US was first performed on the tumor-
bearing mice (Fig. 7). The images were recorded digitally by over-
laying the US enhanced signal (green color) on the background and
analyzed by a small animal US imaging system (Prospect, S-Sharp).
When HSNSs@(DTPA-Gd)-RGD were intravenously administered
into PC3 tumor-bearing mice (tumor target group), the contrast in
the tumor increased significantly (Fig. 7a2). The signal slightly
increased when injected into HeLa tumor-bearing mice (tumor
control group) (Fig. 7b2), whereas there was little increase when
HSNSs@(DTPA-Gd)-PEG (material control group) were adminis-
tered into PC3 tumor-bearing mice (Fig. 7c2). For quantitative
analysis the enhancement of the signal in tumor region, the com-
plete cross-section of the tumor was chosen as a region of interest
for the measurement, and the differences of average image in-
tensity determined by subtraction of the pre- and post-
administration images were automatically calculated (Fig. 7a3, b3,
c3). The inflow of the spheres was recorded for 1 min beginning at
the start of injection. The signal intensity of all groups in the tumor
region increased during the first 10 s after injection completed, and
then maintained at a steady level that revealed the continuous
circulation of the HSNSs in vivo. The signal intensity in the PC-3
tumor region (Fig. 7ac) shown obviously stronger than the tumor
control groups (2 times, Fig. 7b3) and the materials control group (5
times, Fig. 7c3). These results demonstrated that HSNSs@(DTPA- Fig. 8. (a) T1-weighted MR images of tumors at different time (RGDþ/PC3:
Gd)-RGD could be used for targeting real-time monitor of tumor HSNSs@(DTPA-Gd)-RGD were intravenously administered into PC3 tumor-bearing
by US imaging. mice, RGDþ/HeLa: HSNSs@(DTPA-Gd)-RGD were intravenously administered into
Although US imaging can provide real-time information of tu- HeLa tumor-bearing mice, RGD/PC3: HSNSs@(DTPA-Gd)-PEG were intravenously
administered into PC3 tumor-bearing mice). Red circles point out the tumor region. (b)
mor, it still suffers from limited clarity of the tissues. To overcome
The relative signal intensity analysis for T1-weighted MR images of tumors (n ¼ 3,
this drawback, MR imaging was further investigated using *P < 0.05, **P < 0.01). (For interpretation of the references to color in this figure legend,
HSNSs@(DTPA-Gd)-RGD. As shown in Fig. 8a, tumor-bearing mice the reader is referred to the web version of this article.)
were investigated using T1-weighted MR imaging. The MR images
at 1 h post-injection show significant brightness compared to the
image without injection. The tumor site was brighter at 4 h post- agent in vivo. These findings indicated that HSNSs@(DTPA-Gd)-RGD
injection, which indicates time-dependent brightening in the tu- exhibited both positive T1-weighted and US contrast enhancement
mor. This is presumably because the HSNSs do not enter into the in the tumor lesion in vivo, and can be used as a targeted dual-
tumor site completely within a short time. The brightest modality molecular imaging contrast agent for US and MR imaging.
enhancement effect in the tumor was found for the targeted group
(RGDþ/PC3) followed by the tumor control group (RGDþ/HeLa), 3.7. Biodistribution
while T1-weighted MR images of tumor in the material control
group (RGD/PC3) were poor, that consist with coronal plane im- Despite the promising US and MR imaging applications of the
ages (Fig. S5). HSNS@(DTPA-Gd)-RGD, their biodistribution and toxicity in living
The relative MR signal intensity in tumor was further quanti- body is a major concern before them full potential could be
tatively analyzed by ImageJ software (Fig. 8b, compared with the completely realized in diagnose. To understand their circulation
soft tissue). For the targeted group, a significant increase of tumor pathway in the body, we investigated the quantification of the Gd
lesion intensity to 51% was observed 1 h after intravenous injection content in main organs, tumor, and blood by means of ICP-AES.
compared with the MR image before injection, and sustained grew After the mice were injected with HSNS@(DTPA-Gd)-RGD or
with the extension of time. In contrast, only slight MR signal HSNS@(DTPA-Gd)-PEG through the tail vein, various organs and
enhancement was observed in the cell control group (20%) and tissues were collected at 1, 4, and 8 h post-injection for ex vivo
material control group (15%) after injection. The increase in MR studies (Fig. 9). For RGD ligand-specific targeted PC3 tumors, an
signal in both control groups was due to the passive uptake and intense increase of the HSNSs@(DTPA-Gd)-RGD was observed
retention (i.e., enhanced permeability and retention) effect of the exclusively in the liver, lung, and tumor after administration, con-
tumor [52,53]. These observations suggest that HSNSs@(DTPA-Gd)- firming that HSNSs@(DTPA-Gd)-RGD were rapidly taken up by
RGD could be used as a targeted T1-weighted MR imaging contrast these three tissues after injection. Uptake in the heart, kidney, and
L. An et al. / Biomaterials 35 (2014) 5381e5392 5389

Fig. 9. The biodistributions of Gd content in main organs, tumor, urine and feces of mice at 1, 4 and 8 h after intravenous injection of HSNSs. (a) HSNSs@(DTPA-Gd)-RGD injected
into PC3 tumor bearing mice; (b) HSNSs@(DTPA-Gd)-RGD injected into HeLa tumor bearing mice; (c) HSNSs@(DTPA-Gd)-PEG injected into PC3 tumor bearing mice; (d) TEM
micrograph of feces sample at 8 h post-injection after digestion with mixed acids.

spleen was very low in RGDþ/PC3 group (Fig. 9a). The tumor and The distribution results confirmed the uptake of HSNSs predomi-
liver showed the highest uptake of Gd at 4 h post-injection and nantly by the liver and lung. Unfortunately, the retention of HSNSs
then decreased uptake at 8 h, consistent with data obtained from by lung was evident. The Gd uptake in tumor significantly de-
the in vivo MRI images. The lung exhibited the highest uptake of Gd creases because of weak binding between the RGD ligand and tu-
at 1 h and retained a stable level until 8 h, demonstrating that mor cells or no targeting ligands. The distribution trend of the
HSNSs@(DTPA-Gd)-RGD were not easily removed from the lung HSNSs in other main organs and metabolites at different times
within a short period. No uptake increase was detected in blood, post-injection is approximately similar to that of the RGDþ/PC3
confirming the rapid clearance of HSNSs@(DTPA-Gd)-RGD from the targeted group.
circulating blood. Early accumulation in the liver is expected to be Based on the analysis of biodistribution and excretion results,
related to the clearance of the HSNSs@(DTPA-Gd)-RGD from the HSNSs@(DTPA-Gd)-RGD mainly accumulated in the liver, lung, and
blood by the cells of the mononuclear phagocytic system, while tumor, then were gradually metabolized by the liver, and primarily
accumulation of HSNSs@(DTPA-Gd)-RGD in the lung is proposed to eliminated via the feces. Of course, small amounts of
result from their agglomeration caused by the adsorption of plasma HSNSs@(DTPA-Gd)-RGD were likely eliminated by renal excretion.
proteins [54,55]. The above findings indicate that the HSNSs@(DTPA-Gd)-RGD pro-
We also investigated the presence of HSNSs@(DTPA-Gd)-RGD in vide a facile method for dual-modality targeted bio-imaging in vivo
the urine and feces to study their excretion pathway in the mice by and easy quantification of the distribution.
quantitative measure of Gd content. The amount of Gd in feces
increased over time, while the accretion of Gd content in urine 3.8. In vivo toxicity
appeared at 4 h post-injection and then decreased. The Gd content
in feces exhibited higher values compared with that in urine. To Nanoparticles may induce an inflammatory response and in-
directly prove that the HSNSs@(DTPA-Gd)-RGD in urine or feces, crease or decrease the activity of the immune system and alter
the samples were investigated by TEM. Taking the feces sample at related hematological factors [56]. It is necessary to investigate the
8 h post-injection as example, the HSNSs@(DTPA-Gd)-RGD was lesions or inflammation of mice after injection of the HSNSs that
clearly observed as shown in Fig. 9d. These results demonstrated could help us to assess the in vivo toxicity of our dual-model CAs.
that the HSNSs@(DTPA-Gd)-RGD could been eliminated via urine Fluctuation in body weight is an important indicator for studying
and feces. in vivo toxicity effects. The body weights of the mice recorded for 4
The data for the tumor control (RGDþ/HeLa) and material con- weeks were analyzed as shown in Fig. 10a. The change in the body
trol (RGD/PC3) groups are shown in Fig. 9b and c for comparison. weights of mice injected with HSNSs@(DTPA-Gd)-RGD shown no
5390
L. An et al. / Biomaterials 35 (2014) 5381e5392
Fig. 10. (a) Variations in the body-weight of the mice injected with the HSNSs@(DTPA-Gd)-RGD (n ¼ 3, dose ¼ 16 mg/kg, Test) and without injection (n ¼ 3, Control). (bed) Blood analysis results obtained from mice treated with
HSNSs@(DTPA-Gd)-RGD at 1, 4, 8, 24 h and 10 days post-injection (n ¼ 3, dose ¼ 16 mg/kg, Test), and mice receiving no injection (n ¼ 3, Control); (b) The blood levels of liver function index (ALT, ALP and AST); (c) The kidney function
index in the blood: BUN levels (c1), A/G rations (c2), and CREA (c3); (d) The systematic blood analysis data: white blood cells (d1), red blood cells (d2), hemoglobin (d3), hematocrit (d4), mean corpuscular volume (d5), mean
corpuscular hemoglobin (d6), mean corpuscular hemoglobin concentration (d7), and platelets (d8). All the parameters of blood analysis fell well in the normal range. No significant difference in all blood test data was noticed between
control and test groups.
L. An et al. / Biomaterials 35 (2014) 5381e5392 5391

difference with the control group without injection, indicating that Education Institutions of Shanghai (B913113001020) and the Pro-
HSNSs@(DTPA-Gd)-RGD had no side effects on the growth of mice. gram of Shanghai Normal University (DZL124, SK201328).
Once the nanoparticles leave the blood stream and reach the
liver and kidneys, it is important to measure whether or not the
Appendix A. Supplementary data
particles themselves or their constituent materials can induce
toxicity. The systematic blood analysis and H&E staining were
Supplementary data related to this article can be found online at
further studied from the mice treated with HSNSs@(DTPA-Gd)-RGD
http://dx.doi.org/10.1016/j.biomaterials.2014.03.030.
at 1, 4, 8, 24 h and 10 days post-injection (n ¼ 3, dose ¼ 16 mg/kg).
The mice without injection were used as control. As shown in
Fig. 10b, the three hepatic indicators, ALT, ALP and AST were at References
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