Dynamic Article LinksC<Nanoscale

Cite this: Nanoscale, 2011, 3, 4150

www.rsc.org/nanoscale PAPER

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online / Journal Homepage / Table of Contents for this issue

Highly biocompatible TiO2:Gd3+ nano-contrast agent with enhancedlongitudinal relaxivity for targeted cancer imaging†

Parwathy Chandran, Abhilash Sasidharan, Anusha Ashokan, Deepthy Menon, Shantikumar Nairand Manzoor Koyakutty*

Received 9th June 2011, Accepted 6th July 2011

DOI: 10.1039/c1nr10591d

We report the development of a novel magnetic nano-contrast agent (nano-CA) based on Gd3+ doped

amorphous TiO2 of size �25 nm, exhibiting enhanced longitudinal relaxivity (r1) and magnetic

resonance (MR) contrasting together with excellent biocompatibility. Quantitative T1 mapping of

phantom samples using a 1.5 T clinical MR imaging system revealed that the amorphous phase of

doped titania has the highest r1 relaxivity which is �2.5 fold higher than the commercially used CA

Magnevist�. The crystalline (anatase) samples formed by air annealing at 250 �C and 500 �C showed

significant reduction in r1 values and MR contrast, which is attributed to the loss of proton-exchange

contribution from the adsorbed water and atomic re-arrangement of Gd3+ ions in the crystalline host

lattice. Nanotoxicity studies including cell viability, plasma membrane integrity, reactive oxygen stress

and expression of pro-inflammatory cytokines, performed on human primary endothelial cells

(HUVEC), human blood derived peripheral blood mononuclear cells (PBMC) and nasopharyngeal

epidermoid carcinoma (KB) cell line showed excellent biocompatibility up to relatively higher doses of

200 mg ml�1. The potential of this nano-CA to cause hemolysis, platelet aggregation and plasma

coagulation were studied using human peripheral blood samples and found no adverse effects,

illustrating the possibility of the safe intravenous administration of these agents for human

applications. Furthermore, the ability of these agents to specifically detect cancer cells by targeting

molecular receptors on the cell membrane was demonstrated on folate receptor (FR) positive oral

carcinoma (KB) cells, where the folic acid conjugated nano-CA showed receptor specific accumulation

on cell membrane while leaving the normal fibroblast cells (L929) unstained. This study reveals that the

Gd3+ doped amorphous TiO2 nanoparticles having enhanced magnetic resonance contrast and high

biocompatibility is a promising candidate for molecular receptor targeted MR imaging.

Introduction

Targeted molecular imaging agents can enhance the potential of

non-invasive medical imaging from the current scenario of simple

anatomical descriptions to functional in vivo mapping by the

recognition of unique cell surfacemarkers.1Non-invasive detection

of molecular biomarkers can allow early stage diagnosis, better

understanding of the functional characteristics of disease, predic-

tion of prognosis and treatment efficacy analysis leading to signif-

icant improvement in the total diseasemanagement. Recently, with

thedevelopmentofhigh-fieldmagnets and targeted contrast agents,

molecular imaging using magnetic resonance becomes very

popular. This emerging area could be benefitted with the

Amrita Centre for Nanosciences and Molecular Medicine, Amrita Instituteof Medical Sciences and Research Centre Amrita, Vishwa VidyapeethamUniversity, Cochin, 682 041, Kerala, India. E-mail: manzoork@aims.amrita.edu; Fax: +91 484 2802030; Tel: +91 484 4008750

† Electronic supplementary information (ESI) available: FTIR dataGTN-RT and GTN-500. See DOI: 10.1039/c1nr10591d

4150 | Nanoscale, 2011, 3, 4150–4161

development of biocompatible, target specific nano-contrast agents

(nano-CA) that can enhance the signal strength from a particular

receptor or bio-chemical event at molecular level.2 Currently used

CAs for MRI such as Gd3+ complexed with diethyltriamine-pen-

taacetic acid (Gd-DTPA) or tetra azacyclododecane-1,4,7,10-tet-

raacetic acid (Gd-DOTA) are broadly non-specific.3,4

With the advancement of nanotechnology, novel nano-CA

gained increased attention due to their distinct advantages over

Gd-DTPA systems. One of the most important features of nano-

CA is the possibility of targeting them to specific molecular

markers of disease, rendering much better contrast.5 Moreover,

unlike Gd-DTPA having very short blood circulation life time

(�20 min), the pharmacokinetic parameters of nano-CA such as

blood circulation half-life, dwell time at the targeted site etc. can

be controlled by modulating their size, shape or surface chem-

istry.6 Further, due to the presence of large number of para-

magnetic ions (e.g. Gd3+) confined within a nano-scale region,

high rate of proton relaxivity and better image contrast are

expected.

This journal is ª The Royal Society of Chemistry 2011

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

One of the most studied and clinically approved nano-CA is

super-paramagnetic iron oxide (SPIO), which provide high

transverse relaxation (T2) and hence negative contrast.7,8

Compared to paramagnetic Gd3+ based T1 CA (Gd-DTPA) that

provides bright contrast, the dark regions formed by the signal

decreasing effect of superparamagnetic T2 agents could be

confused with other pathogenic conditions such as bleeding,

calcinations, etc.3,9 Moreover, high susceptibility of the T2 CA

induces distortion of the magnetic field on neighboring normal

tissues, whereas, the bright contrast provided by T1 agents

maximizes the forte of MRI with high spatial resolution.

Furthermore, the bright signal can be easily distinguished from

other pathogenic or biological conditions and hence T1 CA are

widely used in the clinics.

A recent review by Hyeon et al.10 discuss emerging trends in

the arena of paramagnetic nano-CA. Mostly, nanostructured

carrier systems that have many anchoring sites for paramagnetic

Gd3+ ions are reported. Various nano-platforms such as silica,11

carbon nanotubes,12 perfluorocarbon emulsions,13 etc have been

used as a carrier for Gd-DTPA and showed the potential

application in MR imaging. Park et al. synthesized gold nano-

particles (Au NPs) coated with Gd-DTPA-bis (amide) conjugate

of glutathione (GdL) as highly efficient MRI CA.14 Similarly,

Paunesku et al. used titanium dioxide (TiO2) nanoparticles to

anchor Gd-DTPA complex on its surface and achieved �1000

fold increase in concentrating paramagnetic ions, 48 h after

delivery.15 Very recently, Jeyarama et al. reported that by

confining Gd based CA within nanoporous structure of silicon

microparticles, multifold enhancement in the r1 relaxivity can be

achieved.16 This report clearly demonstrates the potential

advantage of nano-confinement of CA compared to conven-

tional free systems. Phase pure gadolinium oxide (Gd2O3)17 and

gadolinium phosphate (GdPO4)18 nanoparticles have also been

investigated as MRI CA. They exhibited signal-enhancing

contrast in T1-weighted images with low r2/r1 values. However,

being a 100% gadolinium compound with no surface protecting

layers, pure Gd nanoparticles may cause significant heavy-metal

toxicity.

In contrast to the above approaches, here, we present the

development of a novel nano-CA by doping a low concentration

of Gd3+ ions (max. �2 mol %) into a biocompatible nano-TiO2

matrix. At optimized doping levels, amorphous titania showed

enhanced T1 relaxivity and bright MR contrast compared to the

commercial agent or its crystalline counter parts. The biocom-

patibility of this new nano-CA was studied extensively using

a number of cytotoxicity assays including cell viability, plasma

membrane integrity, pro-inflammatory cytokine expression,

reactive oxygen stress, hemolysis, platelet activation, platelet

aggregation, plasma coagulation test, etc. The cancer targeting

ability of the nano-CA was also demonstrated in folate receptor

expressing cell lines.

Fig. 1 (a) XRD spectra of gadolinium doped TiO2 (GTN) samples; as

prepared (RT), annealed at 250 �C and 500 �C (b) Dynamic light scat-

tering data of as prepared GTN sample showing major (90%) size

distribution �20–25 nm (c) AFM image of as prepared GTN sample

(scale 500 nm) (d) SEM image of GTN sample annealed at 500 �Cshowing spherical nanoparticles of increased size �200 nm.

Results and discussion

Characterization of the GTN

Phase purity and crystallinity of the nanoparticles were analyzed

using X-ray diffraction (XRD) method. Fig. 1a shows diffraction

peaks for as prepared and annealed (250 �C and 500 �C) samples.

This journal is ª The Royal Society of Chemistry 2011

Appearance of characteristic X-ray reflections from (101), (004),

(200), (105), (211) and (213) crystal planes of anatase TiO2

(JCPDS File No. 21–1272) can be seen with increased annealing

temperature while the as prepared sample remain largely amor-

phous. Lattice parameters calculated from the observed X-ray

diffractions matches very well with those of anatase phase having

a ¼ b ¼ 3784 �A and c ¼ 9510 �A. DLS analysis suggested that

more than 90% of the as prepared nanoparticles are within 20–25

nm size range (Fig. 1b). Further, atomic force microscopy of the

sample (Fig. 1c) prepared by spray coating of the colloidal

suspension on an atomically flat mica substrate showed forma-

tion of well dispersed nanoparticles with spherical morphology.

Upon air annealing at 500 �C, this sample showed further crystal

growth leading to the formation of bigger nanoparticles of size

�200 nm, but retaining the spherical shape and monodispersity

as evident from the scanning electron microscopic image

(Fig. 1d).

To confirm the successful doping of Gd3+ and quantitative

estimate of its concentration, GTN samples were subjected to

inductively coupled plasma (ICP) analysis. Interestingly, when 5

and 10 at % of Gd3+ (w.r.t. Ti4+) were used in the reaction

medium, we could detect �2 mol % Gd3+ in the final washed

product in both cases, suggesting that there was no increase in the

doping concentration with increasing Gd3+ above 5 at % in

solution.

Magnetic resonance imaging (MRI)

As the main objective of this study is to develop a T1 weighted

nano-CA, we have studied the MR imaging properties of Gd3+

doped titanium dioxide using phantom experiments. Three

different concentrations; 1, 5 and 10 mg ml�1 of GTN samples

equivalent to Gd3+ concentration of 0.039, 0.195 and 0.39 mg

ml�1 (6.34, 31.7 and 63.4 mM) were dispersed in de-ionized water

in a 96 well plate 250 ml per well). Interestingly from Fig. 2a,

compared to water, Gd(NO3)3 or undoped TiO2, unannealed

GTN sample showed excellent T1 contrast and the signal

intensity increased in a concentration dependent manner.

Nanoscale, 2011, 3, 4150–4161 | 4151

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

Whereas, in case of annealed samples, GTN-250 showed

concentration dependant increase in signal intensity, registering

highest intensity at 5 mgml�1 than 1 mgml�1. However, when the

GTN-250 concentration was further increased to 10 mg ml�1 the

signal intensity reduced drastically resulting in loss of contrast,

probably as a result of the shortening of the T2 relaxation time

(or enhanced r2 effect) due to the increased concentration of Gd3+

ions. On further annealing at 500 �C (GTN-500), there was

almost complete loss of T1 signal for all the concentrations

suggesting that longitudinal relaxivity is severely affected due to

annealing. Relaxivity plot (Fig. 2c) shows that GTN sample

registered highest relaxivity, r1 � 12.25 Mm�1 s�1 which is

�3 fold higher than that of commercial agent Magnevist�(4.1 mM�1 s�1). More interestingly, upon air annealing, GTN-

250 and GTN-500 registered r1 values well below Magnevist�.

This means that, there are significant changes in the proton

exchange rates of Gd3+ situated in an amorphous versus crys-

talline host matrix.

As unannealed GTN showed highest relaxivity, we have

chosen this sample for cell imaging. Fig. 2d shows T1-weighted

images of KB cell pellets incubated with varying concentrations

of GTN (0.2, 0.5, 1 and 5 mg ml�1 equivalent to Gd3+ concen-

tration of 1.2, 3, 6.34 and 31.7 mM) for 24 h. The cell pellets

showed excellent T1 contrast for all concentrations compared to

the control cells.

The observed effect of annealing on the relaxivity of GTN

samples can be explained by considering multiple factors.

Theoretically, the longitudinal relaxivity of a contrast agent can

be enhanced by increasing the number of water molecules in the

first coordination sphere, decreasing the rotational correlation

time (s), and/or by faster water exchange rates.25,26 In the present

case, the un-annealed sample (GTN) showed highest relaxivity

which subsequently decreased with annealing temperature. We

have carefully examined the FTIR spectrum (Supplementary

Fig. 1)† and found that the vibrational signature of water content

(O–H bending and stretching vibrations at 1640 cm�1 and

Fig. 2 (a) T1 weighted phantom NMR images of undoped TiO2 (TN),

Gd3+ doped TiO2 (GTN), GTN air annealed at 250 �C (GTN-250) and

GTN air annealed at 500 �C (GTN-500). Water and 0.1 mM Gd(NO3)3are taken as the controls (b) MR signal intensity plot for different

samples (c) Longitudinal relaxivity plot of samples in comparison to

Magnevist� (d) T1 weighted NMR images of GTN labelled KB cell

pellets acquired in the horizontal plane using pulse sequence spin-echo

technique.

4152 | Nanoscale, 2011, 3, 4150–4161

3200–3400 cm�1) in the GTN sample is drastically reduced on

annealing to 250 �C or 500 �C. This suggests that the change inthe relaxivity is most probably correlated with the loss of water

molecules in the first coordination sphere. In another aspect,

changes in the crystalline nature also might have influenced the

proton exchange rates. XRD studies shows that, GTN sample

giving the best T1 contrast is amorphous in nature whereas with

increasing crystallinity the contrast was lost. We believe that, in

the as prepared sample, most of the Gd3+ ions are doped within

the surface region of the host lattice and there is greater possi-

bility for these Gd3+ ions to have easy proton exchange with the

absorbed water. With annealing, in addition to loss of these

water molecules, atomic re-arrangement of Gd3+ ions within the

lattice may also leads to better coordination with the

surrounding O2� ions and thereby altering the proton exchange

with water rendering lower contrast. In effect, our studies suggest

that, Gd3+ doped amorphous TiO2 can provide high longitudinal

relaxivity and excellent MR contrast compared to its crystalline

counterpart or commercial contrast agents which was further

confirmed through cell pellet imaging experiments.

It is noteworthy that lower concentrations (0.2 mg ml�1) of

GTN itself gave intense T1-weighted MR signal which was

retained satisfactorily in oral cancer cells even after 24 h, without

causing any deleterious effects on cellular viability. This result

raises the possibility of using GTN for targeted (e.g., folate tar-

geted) MR imaging.

Nanotoxicity studies

Nanotoxicity is a major concern which limits the clinical use of

most of the nano-CA reported so far. Although there are plenty

of literature regarding the cytotoxic effects of TiO2 nano-

particles, recent evidences clearly demonstrated better biocom-

patibility of titania compared to other materials.27–30 One of the

latest work conclusively showed that being an insoluble metal

oxide, TiO2 do not cause any serious toxicity effects compared to

relatively soluble materials like ZnO.31 Our recent work on cell-

material interactions of TiO2 nanostructures of distinctly varied

morphologies such as nano-tubes, nano-needles, nano-flowers or

octahedral bipyramidal nanostructures of TiO2, formed on Ti

based bone-implants also showed excellent bio-compatibility and

favorable cell response.32 However, there exist no specific studies

on the nanotoxic effects of Gd3+ doped TiO2 nano-CA, partic-

ularly from the perspective of its application as an intravenously

injectable CA. For such a system, it is essential to carry out

studies on the hemocompatibility and inflammatory response in

human primary cells. Thus, by considering the potential utility of

amorphous GTN as an MR CA, we have employed extensive

toxicity studies analyzing mitochondrial activity, plasma

membrane integrity, intracellular ROS, inflammatory response

and hemocompatibility.

Cytotoxicity studies

Cytotoxicty of undoped (TN) and Gd3+doped titania (GTN)

were carried out in HUVEC and KB cells. HUVEC is a repre-

sentative human primary cell system for toxicity studies consid-

ering the intravenous administration of the nanoparticles in

human applications where the injected nanoparticles are first

This journal is ª The Royal Society of Chemistry 2011

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

exposed to blood cells and primary endothelial cells and hence

the associated toxicity data critical.33 Studies are also done in

cancer cell lines considering the possibility of using GTN for

targeted cancer imaging. Another aspect of this study was to

differentiate between the toxicity of TN and GTN under dark

and light conditions, considering the fact that being a photo-

catalyst, titania could generate reactive oxygen species (ROS)

under light irradiation.34 Fig. 3 shows the cell viability data for

HUVEC and KB cells under dark and light conditions. Inter-

estingly, both the TN and GTN samples showed no toxicity in

dark or light conditions even at relatively higher concentration of

200 mg ml�1. Although primary cells are highly sensitive to

exogenous stress such as ROS, our results showed that TN and

GTN nanoparticles had no significant effects on their viability.

We have extended this study to other cell functions such as effects

on plasma membrane integrity and level of reactive oxygen

stress.

Fig. 4 Lactate dehydrogenase release assay (LDH) of HUVEC and KB

cells treated with TN and GTN samples.

Plasma membrane integrity studies

Integrity of plasma membrane is a cellular trait commonly used

to determine the consequence of physical interactions of nano-

materials with the cell. Changes in the metabolic activity are

indicators of early cell injury, while the effects on membrane

integrity are indicative of more serious injury leading to necrotic

cell death. To investigate this, HUVEC and KB cells were

incubated with TN and GTN samples for 24 h and the culture

supernatants were analyzed for the amount of lactate dehydro-

genase (LDH) enzyme released into it, which is a highly sensitive

and accurate marker for cellular toxicity. Fig. 4a shows that after

24 h incubation, the treated cells showed no sign of LDH leakage

suggesting intact membrane integrity. This supports the MTT

data and further point towards the non-toxic aspect of the

synthesized nanoparticles.

Intracellular ROS stress by flow cytometry

Generation of reactive oxygen species was reported to be one of

the primary reasons for the toxicity of nanomaterials especially

TiO2 type semiconductor metal oxides.35,36 Kocbek et al.31

Fig. 3 MTT data showing non-toxicity of undoped (TN) and Gd3+ doped TiO

of incubation, under light and dark conditions.

This journal is ª The Royal Society of Chemistry 2011

reported a comprehensive toxicological evaluation of TiO2 and

ZnO nanoparticles and confirmed that the latter triggered severe

cytotoxic effects due to ZnO dissolution whereas TiO2 nano-

particles remained more or less inert. Similar to MTT assay, we

have conducted this study both under dark and light conditions,

as there is a greater possibility for TiO2 to release ROS under

light. Results (Fig. 5) suggest that even under light irradiated

conditions, TN and GTN (200 mg ml�1, 24 h incubation) hardly

produced any intracellular ROS stress. This suggests that GTN

nano-CA prepared though our method impart no toxicity asso-

ciated with ROS stress.

2 (GTN) to human primary cell (HUVEC) and cancer cell (KB) after 24 h

Nanoscale, 2011, 3, 4150–4161 | 4153

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

Inflammation studies: Quantitative analysis by flow cytometry

One of the major hurdles for an intravenously injected nano-CA

will be to overcome immune/inflammatory response, which may

lead to premature clearance of agents from the blood pool.37

During a typical inflammatory response, leukocytes accumulate

at the site of damage after trans-endothelial migration, mediated

by a cascade of events involving release of pro-inflammatory

cytokines, chemokines and adhesion molecules. The major

mediators of these processes are IL-1b and TNF-a which are

produced mainly by activated monocytes/macrophages which, in

turn, activate these cells. Other immediate cytokines involved in

inflammation include IL-6, IL-8, IL-10 and IL-12. Using flow

cytometry and fluorescent beads conjugated to respective anti-

bodies we have quantitatively measured the levels of IL-1b, IL-6,

IL-8, IL-10, TNF-a and IL-12p70 in human peripheral blood

derived mononuclear cells (PBMC) treated with 10 and 50 mg

ml�1 TN and GTN, for 24 h. As shown in Fig. 6a, TN and GTN

did not induce any significant expression of IL-1b, IL-8, IL-10,

IL-12p70 or TNF except for IL-6 which showed relatively higher

expression (24%), but only at 50 mg ml�1. IL-6 is an interleukin

that acts both as a pro-inflammatory and anti-inflammatory

cytokine secreted by T-cells and macrophages to stimulate

immune response to trauma, especially burns or other tissue

damage, leading to inflammation. IL-6’s role as an anti-inflam-

matory cytokine is mediated though its inhibitory effects on

TNF-a and IL-1b, and activation of IL-1RA and IL-10. Our

samples did not express TNF-a or IL-1b, which might point

towards the activation of its anti-inflammatory role, rather than

its pro-inflammatory side. The untreated control cells were

shown to express slightly high level of IL-8 than normal, which

might have resulted from the stress exerted during their isolation

and culturing. From the above data, it can be seen that TN and

GTN cause minimal inflammation in the human system.

Blood contact studies

Another important nanotoxicity aspect of an intravenously

administered CA is its hemocompatibility. We have used

a battery of blood compatibility tests including hemolysis,

Fig. 5 Flow cytogram showing intracellular ROS generation in GTN treated

Control cells showing no residual ROS (b) Cells treated with 300 mM H2O2 s

GTN showing insignificant ROS generation.

4154 | Nanoscale, 2011, 3, 4150–4161

platelet aggregation and plasma coagulation to evaluate the

hemocompatibility of the TN and GTN samples.

Hemolysis

Hemolysis is the process where the red blood cells (RBC) rupture

leading to leakage of hemoglobin. Mostly, nanoparticle induced

hemolysis is contributed by the size, shape (needular structures),

surface charge or composition of the material.31 We have tested

for such possible effects by treating RBC with varying concen-

trations (10, 50, 100, 150 and 200 mg ml�1) of TN and GTN

samples. Interestingly, Fig. 7a shows no hemolysis even up to

high concentration of 200 mg ml�1 nanoparticle treated samples

compared to the positive control (1% Triton X-100) registering

�100% hemoglobin leakage. This is also evident from the optical

image shown in Fig. 7b where the Triton treated sample showed

significant leakage of hemoglobin into the plasma supernatant

while 200 mg ml�1 GTN treated sample showed undamaged RBC

sedimented to the bottom of the microcentrifuge tube, leaving

clear plasma above. The result was further confirmed using

scanning electron microscopic imaging of the GTN (200 mg ml�1)

treated RBC (Fig. 7c) where we can clearly see the intact

morphology of the RBC even though large concentration of

nanoparticles are adhered on to its cell membrane. This confirms

the non-hemolytic property of GTN nano-CAs.

Platelet aggregation and plasma coagulation studies

Platelet aggregation analysis is relevant to understand whether

the nano-CA triggers the coagulation cascade by platelet acti-

vation leading to platelet aggregation. Our results shown in

Fig. 8a suggest that both TN and GTN samples hardly induced

any apparent change in platelet count indicating that platelet

aggregation was not initiated. Fig. 8b and 8c shows the SEM

images of untreated platelets (negative control) and ADP treated

platelets (positive control) respectively. As seen in Fig. 8b the

untreated platelets spread on the poly-L-lysine coated coverslips

without any aggregation. Fig. 8c shows the SEM image of ADP

treated platelets, where agglomerated platelets could be seen

spreading on the coverslip.

HUVEC cells using dichlorofluoroscein-diacetate (DCFH-DA) assay: (a)

howing high levels of ROS generation (c) Cells treated with 200 mg ml�1

This journal is ª The Royal Society of Chemistry 2011

Fig. 6 (a) Flow cytogram depicting the inflammatory profile of the nanoparticles in comparison with the controls (b) Graph showing the activation of

various cytokines by 50 mg ml�1 TN and GTN samples.

Fig. 7 (a) In vitro hemolysis study on RBC treated with varying

concentrations of TN and GTN samples. Both samples showed no

hemolysis even up to high concentration of 200 mg ml�1 (b) Optical image

of RBC treated with 200 mg ml�1 GTN sample showing no hemolysis

whereas 1% Triton X-100 (positive control) treated RBC showing leakage

of hemoglobin to the supernatant (c) SEM photograph of GTN treated

(200 mg ml�1 for 3h) RBC showing intact cell morphology.

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

Possibility of the developed nano-CA to interfere with the

normal coagulation time was analyzed using two tests, Pro-

thombin Time (PT) test and Activated Partial Prothombin Time

(aPTT) test. For both PT and aPTT analysis platelet poor

plasma (PPP) was obtained from freshly drawn blood and

incubated with samples at 37 �C for 30 min. Prothombin time

(PT) analysis is done to study the effect of the test material on the

activity of factors involved in the extrinsic pathway including

Factor II, V, VII, X, and fibrinogen. PT test is performed to

measure the time taken to form a clot after the sequential addi-

tion of tissue thomboplastin and Ca2+ ions to PPP. The normal

range for PT test is 10 to 15 s depending on the reagent used. The

PT time in sec, obtained for varying concentrations of TN and

GTN samples, are shown in Fig. 9a. The variation in PT time

between different nanoparticle concentrations ideally fell within

This journal is ª The Royal Society of Chemistry 2011

the normal range (highlighted region) indicating that the nano-

particles did not cause any significant interference in the extrinsic

cascade.

Activated partial thomboplastin time (aPTT) is an assay used

to screen for any possible interference with the coagulation

factors of the intrinsic clotting cascade, namely, Factors I, II, V,

VIII, IX, X, XI, XII, prekallikrein and high molecular weight

kininogen (HMWK). An aPTT assay is performed by the addi-

tion of a Factor XII activator, a phospholipid, and Ca2+ ions to

PPP. The normal time range is usually reported to be 25 to 35 s.

aPTT result is usually represented as a ratio, International

Normalized Ratio (INR), which suggests the normal range to be

between 0.9–1.2. Fig. 9b shows the aPTT ratio variation within

the normal range (highlighted in orange) demonstrating that the

nanoparticles did not interfere with the coagulation factors of the

intrinsic pathway.

Folate mediated cancer targeting

Considering the excellent biocompatibility shown by GTN

samples, we have further tested its potential to target folate

receptor positive cancer cells. FR expression on normal tissues is

highly restricted, making it a useful marker for targeted drug

delivery to tumors.38,39 The sequential bio-conjugation scheme is

depicted in Fig. 10a, wherein a thin shell of albumin (BSA)

protein pre-loaded with Rh123 was coated over the GTN and the

shell was further surface derivatized with FA using EDC-NHS

chemistry.40,41 The conjugation steps were analytically followed

by FTIR spectroscopy. Fig. 10b compares the FTIR spectra of

folic acid, BSA and bare GTN with that of GTN-BSA-Rh123-

FA conjugate. GTN sample showed a broad band peak at�3395

cm�1 corresponding to the O–H stretching mode of physically

absorbed water and the peak at �627 cm�1 signifies the Ti-O

stretching mode in amorphous TiO2.19 The characteristic amide I

band of BSA can be seen at 1654 cm�1 as expected for a protein

with a high proportion of a-helix and the band centered at

�3474 cm�1 can be attributed to primary amines. The broad

Nanoscale, 2011, 3, 4150–4161 | 4155

Fig. 8 (a) Platelets treated with TN and GTN showing no detectable change in the count indicating that platelet aggregation is not initiated (b) SEM

photograph showing GTN treated platelets exhibiting intact morphology without any aggregation or activation (c) SEM photograph of adenosine

diphosphate (ADP) treated platelets showing aggregation.

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

band at �704 cm�1 can be attributed to �NH2 and �NH

wagging. FTIR spectrum of GTN-BSA-Rh123-FA shows the

characteristic peaks associated with FA aromatic ring stretching

of the pyridine ring and p-amino benzoic acid of FA in the range

of 1478–1654 cm�1. Broader peak at 3408 cm�1 is related to the

stretching vibration of N–H bond in primary amines and amide

linkage. The line broadening appearing over 1654 cm�1 to

1237 cm�1 is indicative of the covalent linkage of FA with BSA.42

This study confirms successful conjugation of FA to GTN-BSA-

Rh123 conjugate.

Folate receptor targeted delivery of GTN-BSA-Rh123-FA CA to

cancer cells

Finally, we demonstrated the possibility of molecular receptor

targeted delivery and specific imaging of folate receptor positive

(FR+ve) cancer cells using FA conjugated GTN nano-CA. For

this, we used two different cell lines – FR+ve oral cancer (KB) cells

showing high expression of folate receptors and mouse fibroblast

L929 cells showing normal expression level of folate receptors

(Fig. 11), which was characterized by our group.43 Fig. 12a and

12b shows fluorescence microscopic images of L929 treated with

GTN-BSA-Rh123-FA for 1 and 4 h respectively. It is clear from

the figure that even after 4 h of incubation, the conjugated

Fig. 9 (a) Prothrombin time analysis of TN and GTN samples showing no si

to the normal range (highlighted in orange) of 12–15 s (b) Activated partial t

variation within the normal range of 0.9–1.2 (highlighted in orange).

4156 | Nanoscale, 2011, 3, 4150–4161

nanoparticles were randomly distributed all around the cell

without any specific interaction with the cell membrane. Fig. 12c

and 12d shows FR+ve KB cells treated with GTN-BSA-Rh123-

FA for 1 and 4 h of incubation respectively. The nanoparticles

showed specific attachment to the cell membrane even within 1 h,

which was significantly enhanced with prolonged incubation for

4 h. This clearly illustrated the specificity of FA conjugated

nanoparticles in detecting folate receptors on the surface of KB

cells. Thus, we showed that the conjugated GTN based MR

contrast agents can be used for actively targeted cancer imaging.

Conclusion

We report the development of a nano-contrast agent based on

monodispersed, amorphous Gd3+ doped TiO2 nanoparticles

having average size of �25 nm exhibiting excellent MR contrast

properties and biocompatibility. Doping with Gd3+ ions

conferred TiO2 with paramagnetic functionality suitable for T1

weighted MR imaging. Phantom MR images taken with 1.5 T

clinical MR system proved the potential of the material as MR

contrast agent. Nanotoxicity studies including cell-viability,

plasma membrane integrity, reactive oxygen stress analysis and

expression of pro-inflammatory cytokines, performed on human

primary endothelial cells (HUVEC), human peripheral blood

gnificant variation from the negative control. PT time variation is limited

hromboplastin time (aPTT) ratio of TN and GTN samples showing the

This journal is ª The Royal Society of Chemistry 2011

Fig. 10 (a) Schematic diagram depicting the sequential bio-conjugation steps involving coacervation, coating of GTN with Rhodamine 123 (Rh123)

tagged Bovine Serum Albumin (BSA), and further surface derivatization of the GTN-BSA-Rh123 with folic acid (FA) using the EDC-NHS chemistry

(b) FTIR spectra of FA, BSA, GTN and GTN-BSA-Rh123-FA, recorded in KBr supported pellets. The characteristic vibration bands related to BSA

and FA can be clearly seen in the final conjugates of GTN-BSA-Rh123-FA.

Fig. 11 FR expression studies using flow cytometry. (a) FACS results of the FR expression on nasopharyngeal cancer cell line (KB) and normal

fibroblast cell line (L929); upper panel shows the isotype controls and lower panel shows the test samples (b) Mean fluorescence intensity histogram

showing high fluorescence intensity related to anti-FR antibody stained KB cells indicating over-expression of FR.

This journal is ª The Royal Society of Chemistry 2011 Nanoscale, 2011, 3, 4150–4161 | 4157

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

Fig. 12 Fluorescent microscopic images showing interaction of GTN-

BSA-Rh123-FA samples with FR�ve normal fibroblast cell line L929 after

(a) 1 h (b) 4 h of incubation and with FR+ve oral cancer cell line KB after

(c) 1 h (d) 4 h of incubation. Specific attachment of GTN-BSA-Rh123-

FA on KB cell membrane can be clearly seen from the images.

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

mononuclear cells (PBMC) and nasopharyngeal epidermoid

carcinoma (KB) cells showed no apparent toxicity even up to

relatively higher doses of 200 mg ml�1. A battery of blood contact

studies including hemolysis, platelet aggregation and plasma

coagulation, of the GTN samples in human blood derived

peripheral blood mononuclear cells, revealed excellent hemo-

compatibility illustrating the possibility of safe intravenous

administration into human system. The ability of fluorescent

tagged nanoparticles to specifically detect and image molecular

receptors on cell membrane was demonstrated on folate receptor

positive oral carcinoma, KB cells, where the folic acid conjugated

GTN-BSA-Rh123 showed receptor specific aggregation while

leaving the normal L929 cells unstained. GTN with molecular

imaging functionality in accord with biocompatibility and

molecular receptor specific targeting capability of doped TiO2

presents a promising MR CA.

Experimental

Synthesis of GTN nano-CA

For synthesizing Gd3+ doped TiO2 nanoparticles (GTNs), we

have modified the alkoxide route19 and optimized the doping

parameters to obtain magnetic properties suitable for a T1

contrast imaging with enhanced relaxivity for best possible MR

imaging. In a typical process, 3.8 ml of 0.1 M Gd(NO3)3 was

added to 1 ml 3.8 M titanium isopropoxide and 25 ml ethylene

glycol, to obtain�10 at% of Gd3+ dopant ions with respect to Ti4+

ions in the reaction medium. For homogenizing the reaction

precursors, the mixture was stirred for 8 h under nitrogen

purging. A mixture of water and acetone in the ratio 1.58 : 100

was added into the above solution and stirred vigorously for 1 h

which yielded a white colloidal suspension. The white precipitate

was harvested by centrifugation and washed several times with

ethanol and hot water to remove ethylene glycol and unreacted

Gd(NO3)3, respectively. The final washed product was dried at 60�C and this sample is referred hereafter as GTN. A portion of this

powder sample was subjected to mild air annealing at 250 �C

4158 | Nanoscale, 2011, 3, 4150–4161

(GTN-250) and 500 �C (GTN-500). As a control sample for

magnetic resonance imaging studies, undoped titania nano-

particles (TN) were also prepared using the same procedure but

without adding Gd(NO3)3. Both annealed and un-annealed

samples were taken for MR phantom studies.

Characterization

Crystallinity of the samples was studied using a PANalytical X

Pert-Pro X-ray powder diffractometer fitted with Cu-Ka source

(l ¼ 1.541 �A). The phase identification was done using the

standard JCPDS database (JCPDS File No. 21–1272). Average

size distribution of GTN was measured using dynamic light

scattering technique (PSS-NICOMP380 ZLS Particle Size

Analyzer). Particle size and morphology was further confirmed

using Atomic Force Microscopy [JEOL-JSPM-5200, Japan] and

scanning electron microscopy [JSM-6490LA, Japan].

Compositional analysis of synthesized GTN by ICP

Gd3+ content in GTNs was quantified using an Inductively

Coupled Plasma with Optical Emission Spectrometry (Varian

715-ES ICP-OES, USA). GTN samples were prepared by

microwave digestion with ultra-pure concentrated aqua regia

(3HNO3: 1HCl) before analysis in ICP-OES.

Magnetic resonance imaging (MRI) phantom studies

MR imaging experiments were performed with a 1.5 T clinical

scanner (GE Healthcare, USA) with a standard quadrature head

coil for RF transmission and detection. For T1-weighted MR

imaging, doped and undoped TiO2 nanoparticles of different

concentrations (1, 5 and 10 mg ml�1) were dispersed in water and

taken in 96 well plate for imaging. Aqueous dispersion of

undoped titania of same concentration (1, 5 and 10 mgml�1), and

water were taken as negative controls while aqueous Gd(NO3)3solution equivalent to the concentration of Gd3+ ions present in

the GTN samples was taken as positive control. For cell pellet

imaging, 1 � 107 KB cells were incubated with 0.2–5 mg ml�1 of

GTN for 24 h and transferred to 1.5 ml microcentrifuge tubes

and pelleted by centrifugation. Images were recorded by pulse

sequence spin-echo technique adopting the following parameters

for image acquisition: TE ¼ 9 ms, TR ¼ 200 ms, FOV 19 � 19

cm2, resolution 256 � 256 points, band width 41.67 KHz and

number of acquisitions ¼ 4. All experiments were done at room

temperature (25 �C).The r1 relaxivity was measured for 0.6% agar phantoms of

GTN samples of varying Gd3+ concentrations (0–1 mM) using

spin-echo sequence (128� 256, 1 NEX, 20 cm field of view, 6 mm

thick slices). Two sets of scans, one set containing 13 scans with

varying TR (TE¼ 8 ms; TR range: 100–15,000 ms) and the other

set containing 10 scans with varying TE (TR ¼ 3000 ms; TE

range: 8–200 ms) were performed. Average signal intensity in

each scan sequence was then obtained from a circular region of

interest (ROI), measured in triplicate and subsequently used to

calculate relaxation times and rates for each concentration.

This journal is ª The Royal Society of Chemistry 2011

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

Cytotoxicity studies

Human umbilical vein endothelial cells (HUVEC) were isolated

from umbilical cord veins of healthy volunteers, as per relevant

literature20 after obtaining approval from the Institutional Ethics

Committee (IEC) of Amrita Institute of Medical Sciences and

Research Centre, Kerala, India. The isolated cells were main-

tained in Iscove’s Modified Dulbecco’s Medium (IMDM; Invi-

trogen, CA, USA) supplemented with 20% Fetal Bovine Serum

(FBS; Invitrogen, CA, USA) and Endothelial Cell Growth

Supplement (ECGS; Sigma, St. Louis, USA). The cells were

subcultured by trypsinization with Trypsin–EDTA, seeded in cell

culture flasks coated with 2% gelatin. Passages 3–4 were selected

for all the experiments. Peripheral blood mononuclear cells

(PBMC) were isolated from human peripheral blood of healthy

volunteers using the Ficoll-Paque density gradient centrifuga-

tion21 after obtaining approval from the IEC. The isolated cells

were re-suspended in 10% RPMI-1640 and used for relevant

experiments. Human nasopharyngeal epidermoid carcinoma

(KB) and mouse fibroblast (L929) cell lines were procured from

National Center for Cell Science, Pune, India. KB and L929 cells

were maintained in Minimal Essential Medium (MEM, Invi-

trogen, CA, USA) supplemented with 10% FBS. All media were

supplemented with 50 IU ml�1 penicillin and 50 mg ml�1 strep-

tomycin. Cells were incubated in a humidified atmosphere of 5%

CO2 at 37�C.

Cell viability assay

MTT (Sigma, St. Louis, USA) assay is used to determine the cell

viability of cells treated with the nano-contrast agents as previ-

ously described.22 Various concentrations (10, 50, 100, 150 and

200 mg ml�1) of undoped (TN) and doped (GTN) nanoparticles

were treated with HUVEC and KB cells for 24 h. 1% Triton

X-100 treated cells served as the positive control and untreated

cells as the negative control.

Plasma membrane integrity assay

Lactate dehydrogenase (LDH) released from the dead and/or

damaged cells were quantified using a commercial assay kit

(Sigma, St. Louis, USA) using manufacturer’s protocol.23

Measurement of intracellular ROS level by flow cytometry

Intracellular generation of reactive oxygen species was deter-

mined using 20, 70 dichlorofluorescein diacetate assay as repor-

ted44 (DCFH-DA; Invitrogen, CA, USA). HUVEC cells were

treated with �200 mg ml�1 GTN for 24 h and retrieved for

DCFH-DA assay using flow cytometry (BD FACS Aria� II,

Becton Dickinson, USA).

Inflammation and hemocompatibility studies

Human inflammation cytokine assay. Cytometric Bead Array

(CBA; BD Biosciences, USA) was used to study the human

cytokine expression (IL-8, IL-1b, IL-6, IL-10, TNF and IL-

12p70 proteins) after nanoparticle treatment. 1� 106 cells/well of

PBMC were treated with 10, 50, 100, 150 and 200 mg ml�1 of

undoped (TN) and doped (GTN) nanoparticles for 24 h. 1 mg

ml�1 lipopolysacccharide (LPS) was used as the positive control,

This journal is ª The Royal Society of Chemistry 2011

while untreated cells as the negative control. After 24 h, the

culture supernatants were analyzed for the expression of the

above mentioned 6 cytokines. Quantification of the cytokines

was done using the equation, % activation ¼ [(Cs – Cn)/(Cp – Cn)

� 100, where Cs, Cn and Cp are the cytokine concentrations

obtained for sample, negative control (medium) and positive

control (LPS) respectively.

Hemolysis. Hemolytic potential of nanoparticles was assessed

using spectrophotometric quantification of free hemoglobin (Hb)

released into blood plasma employing the Soret band absorption

of oxyhemoglobin at 415 nm.24 Freshly drawn blood from

healthy human volunteers was treated with 50 ml of samples for

3 h at 37 �C under mild mixing condition. PBS and 1% Triton

X-100 were used as negative and positive controls respectively.

After incubation plasma was collected and diluted with 0.01%

sodium carbonate and absorbance was measured using spectro-

photometer (UV-VIS 1700 Pharma Spec, Shimadzu, Japan)

Amount of plasma hemoglobin was calculated using equation,

Amount of plasma hemoglobin (mg dL�1)¼ [(2�A415) – (A380 +

A450) � 1000 � dilution factor]/(E � 1.655), where A415, A380,

A450 are the absorbance values at 415, 380 and 450 nm. A380 and

A450 are correction factors applied for uroporphyrin absorption

falling in the same wavelength range of Hb. E is molar absorp-

tivity value of oxyhemoglobin at 415 nm which is 79.46. 1.655 is

the correction factor applied to nullify the interference from the

turbidity of plasma sample. The hemolytic property of different

sample concentrations of nanoparticles was plotted as

percentage hemolysis (% Hemolysis ¼ Plasma Hb of sample/

Total blood Hb) X 100.

Platelet aggregation studies

Platelet aggregation was monitored in platelet rich plasma (PRP)

obtained from freshly drawn human whole blood centrifuged at

150 g for 10 min. 800 ml of PRP was treated with 200 ml of sample

to be tested at 37 �C for 15 min. 50 mM Adenosine diphosphate

(ADP; Sigma, St. Louis, USA) and untreated PRP served as

positive and negative control respectively. Sample treated PRP

was analyzed using a hematology analyzer which determines

number of active platelets. Percent aggregation is calculated by

comparing number of active platelets in a test sample to the one

in control baseline tube.

Plasma coagulation studies

Effect of nanoparticles on plasma coagulation times are assayed

employing two plasma coagulation time measurements i.e. pro-

thombin time (PT) and activated partial thomboplastin time

(aPTT). Platelet poor plasma (PPP) was obtained by centrifu-

gation of whole blood at 4000 rpm for 15 min at 20 �C. 450 ml of

PPP was treated with 50 ml of sample at 37 �C for 30 min. 100 ml

of prothrombin reagent (Diagnostica Stago, France) was added

to 50 ml of treated plasma and the time taken for the plasma to

coagulate was measured as the PT value of the sample. In case of

aPTT measurement, 50 ml of aPTT activator (Diagnostica Stago,

France) was added to 50 ml of plasma and incubated for 180 s.

After the stipulated time, added 50 ml of 0.025MCaCl2. The time

taken by plasma to coagulate after CaCl2 addition was measured

Nanoscale, 2011, 3, 4150–4161 | 4159

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

as aPTT. aPTT value was expressed as aPTT ratio (aPTT of

ssssample/aPTT of control). PT and aPTT values were recorded

in a STArt4 Coagulometer (Diagnostica Stago, France)

according to the operation guidelines detailed by instrument

manufacturer.

Folic acid and Rhodamine123 conjugation for cancer targeted

fluorescence imaging

GTN nanoparticles are coated with a layer of protein, albumin,

using the coacervation method to enable surface derivatization

with folic acid (FA) and to tag a fluorescent dye, Rhodamine 123

(Rh123). EDC-NHS chemistry40 was employed for the FA

conjugation.

Flow cytometric characterization of cells for FR expression

KB and L929 cells were grown in 6 well plates in RPMI medium.

Cells were trypsinized at 80% confluency, counted, and resus-

pended in 100 ml of 1% FBS–PBS. Cells were incubated with 10 ml

of PE conjugated anti-FR a antibody (anti-hFOLR1, R & D

Systems, Minneapolis, USA) for 1 h in the dark. Cells were

washed twice with FBS–PBS to remove excess and weakly bound

antibodies, and analyzed using flow cytometer. Respective iso-

type controls were also analyzed.

Fluorescent microscopic studies for folate receptor targeted

imaging

Cells (KB and L929) were seeded on 13 mm glass cover slips

placed inside 24 well plate at a seeding density of 5000 cells/cover

slip. After 24 h, the adherent cells were washed once with PBS

followed by replacement of media containing 100 mg ml�1 GTN-

BSA-Rh123-FA conjugate and incubated for 1–4 h at 37 �C.Cells were washed once with PBS, fixed with 2% para-

formaldehyde for 20 min and mounted with DPX mounting

medium. Imaging was done using Olympus BX-51 fluorescent

microscope equipped with CCD camera (Model DP71).

Acknowledgements

We thank Department of Biotechnology (DBT), Govt. of India

for the financial support under the project ‘Nanotoxicology and

Nanomedicine’ (BT/PR9357/NNT/28/104/2007). We also thank

Department of Science and Technology (DST), Govt. of India

for the financial support under ‘Nano-Center Grant’ (SR/S5/

NM-51-2005) and ‘Nanotheragnostics’ (SR/NM/NS-99/2009).

Authors also thank Dr Rajesh Kannan, Dept. of Radiology,

Amrita Institute of Medical Science, for MR measurements, Mr.

Sajin P. Ravi for his support in scanning electron microscopy and

Mrs. Sreerekha P. R. for FACS measurements.

References

1 R. Weissleder, Science, 2006, 312, 1168–1171.2 G. M. Lanza, P. M. Winter, S. D. Caruthers, A. M. Morawski,A. H. Schmieder, K. C. Crowder and S. A. Wickline, J. Nucl.Cardiol., 2004, 11, 733–743.

3 H. J. Weinmann, R. C. Brasch, W. R. Press and G. E. Wesbey, Am. J.Roentgenol., 1984, 142, 619–624.

4 D. Baumann and M. Rudin, Magn. Reson. Imaging, 2000, 18, 587–595.

4160 | Nanoscale, 2011, 3, 4150–4161

5 H. B. Na, I. C. Song and T. Hyeon, Adv. Mater., 2009, 21, 2133–2148.6 P. Sharma, S. Brown, G. Walter, S. Santra and B. Moudgil, Adv.Colloid Interface Sci., 2006, 123–126, 471–485.

7 A. Bjørnerud and L. Johansson, NMR Biomed., 2004, 17, 465–477.8 C. C. Berry and A. S. G. Curtis, J. Phys. D: Appl. Phys., 2003, 36,R198–R206.

9 S. Flacke, S. Fischer, M. J. Scott, R. J. Fuhrhop, J. S. Allen,M. McLean, P. Winter, G. A. Sicard, P. J. Gaffney, S. A. Wicklineand G. M. Lanza, Circulation, 2001, 104, 1280–1285.

10 P. M. Winter, A. M. Morawski, S. D. Caruthers, R. W. Fuhrhop,H. Zhang, T. A. Williams, J. S. Allen, E. K. Lacy, J. D. Robertson,G. M. Lanza and S. A. Wickline, Circulation, 2003, 8, 2270–2274.

11 W. J. Rieter, J. S. Kim, K. M. Taylor, H. An, W. Lin, T. Tarrant andW. Lin, Angew. Chem., Int. Ed., 2007, 46, 3680–3682.

12 C. Richard, B. T. Doan, J. C. Beloeil, M. Bessodes, E. T�oth andD. Scherman, Nano Lett., 2008, 8, 232–236.

13 A. M. Neubauer, J. Myerson, S. D. Caruthers, F. D. Hockett,P. M. Winter, J. Chen, P. J. Gaffney, J. D. Robertson,G. M. Lanza and S. A. Wickline, Magn. Reson. Med., 2008, 60,1066–107.

14 J. A. Park, P. A. Reddy, H. K. Kim, I. S. Kim, G. C. Kim, Y. Changand T. J Kim, Bioorg. Med. Chem. Lett., 2008, 18, 6135–613.

15 T. Paunesku, T. Ke, R. Dharmakumar, N. Mascheri, A. Wu, B. Lai,S. Vogt, J. Maser, K. Thurn, B. Szolc-Kowalska, A. Larson,R. C. Bergan, R. Omary, D. Li, Z. R. Lu and G. E. Woloschak,Nanomed.: Nanotechnol., Biol. Med., 2008, 4, 201–207.

16 J. S. Ananta, B. Godin, R. Sethi, L. Moriggi, X. Liu, R. E. Serda,R. Krishnamurthy, R. Muthupillai, R. D. Bolskar, L. Helm,M. Ferrari, L. J. Wilson and P. Decuzzi, Nat. Nanotechnol., 2010, 5,815–821.

17 F. Evanics, P. R. Diamente, F. C. J. M. Veggel, G. J. Stanisz andR. S. Prosser, Chem. Mater., 2006, 18, 2499–2505.

18 H. Hifumi, S. Yamaoka, A. Tanimoto, D. Citterio and K. Suzuki, J.Am. Chem. Soc., 2006, 128, 15090–15091.

19 X. Jiang, T. Herricks and Y. Xia, Adv. Mater., 2003, 15, 1205–1209.20 B. Baudin, A. Bruneel, N. Bosselut and M. Vaubourdolle, Nat.

Protoc., 2007, 2, 481–485.21 D. L. Feldman and T. C. Mogelesky, J. Immunol. Methods, 1987, 102,

243–249.22 T. Mossman, J. Immunol. Methods, 1983, 65, 55–63.23 D. C. Rodney, G. B. John, O. B. Russell and R. L. C. Roger, Clin.

Chem., 1966, 12, 406–413s.24 M. Harboe, Scand. J. Clin. Lab. Invest., 1959, 11, 66–70.25 P. Hermann, J. Kotek, V. Kubicek, I. Lukes and C. Caravan, Dalton

Trans., 2008, 23, 3027–47.26 S. Setua, D. Menon, A. Asok, S. Nair and M. Koyakutty,

Biomaterials, 2010, 31, 714–29.27 D. B. Warheit, R. A. Hoke, C. Finlay, E. M. Donner, K. L. Reed and

C. M. Sayes, Toxicol. Lett., 2007, 171, 99–110.28 T. Xia, M. Kovochich, M. Liong, L. M€adler, B. Gilbert, H. Shi,

J. I. Yeh, J. I. Zink and A. E. Nel, ACS Nano, 2008, 2, 2121–34.29 H. A. Jeng and J. Swanson, J. Environ. Sci. Health Part A, 2006, 41,

2699–2711.30 K. Peters, R. E. Unger, C. J. Kirkpatrick, A. M. Gatti and E. Monari,

J. Mater. Sci.: Mater. Med., 2004, 15, 321–325.31 P. Kocbek, K. Teskac, M. E. Kreft and J. Kristl, Small, 2010, 6, 1908–

1917.32 V. V. Divya Rani, K. Manzoor, D. Menon, N. Selvamurugan and

S. V. Nair, Nanotechnology, 2009, 20, 195101.33 C. F. Jones and D. W. Grainger, Adv. Drug Delivery Rev., 2009, 61,

438–456.34 M. R. Hoffmann, S. T. Martin, W. Choi and D. W. Bahnemann,

Chem. Rev., 1995, 95, 69–96.35 A. Nel, T. Xia, L. M€adler and N. Li, Science, 2006, 311, 622–627.36 A. Sasidharan, P. Chandran, D. Menon, S. Raman, S. Nair and

M. Koyakutty, Nanoscale, 2011, DOI: 10.1039/C1NR10272A.37 (a) M. A. Dobrovolskaia, P. Aggarwal, J. B. Hall and S. E. McNeil,

Mol. Pharmaceutics, 2008, 5, 487–495; (b) M. A. Dobrovolskaia,J. D. Clogston, B. W. Neun, J. B. Hall, A. K. Patri andS. E. McNeil, Nano Lett., 2008, 8, 2180–2187.

38 R. J. Lee and P. S. Low, J. Biol. Chem., 1994, 269, 3198–204.39 K. Manzoor, S. Johny, D. Thomas, S. Setua, D. Menon and S. Nair,

Nanotechnology, 2009, 20, 065102.40 G. T. Hermanson 2008 Bioconjugate Techniques 2nd Edition

Academic Press Inc. 171–172.

This journal is ª The Royal Society of Chemistry 2011

Dow

nloa

ded

by U

nive

rsity

of

Que

ensl

and

on 1

4/04

/201

3 14

:15:

29.

Publ

ishe

d on

18

Aug

ust 2

011

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C1N

R10

591D

View Article Online

41 A. Ashokan, D. Menon, S. Nair and M. Koyakutty, Biomaterials,2010, 31, 2606–16.

42 A. Retnakumari, S. Setua, D. Menon, P. Ravindran, H. Muhammed,T. Pradeep, S. Nair and M. Koyakutty, Nanotechnology, 2010, 21,055103.

This journal is ª The Royal Society of Chemistry 2011

43 S. Narayanan, N. S. Binulal, U. Mony, K. Manzoor, S. Nair andD. Menon, Nanotechnology, 2010, 21, 285107.

44 A. Sasidharan, L. S. Panchakarla, P. Chandran, D. Menon, S. Nair,C. N. Rao and M. Koyakutty, Nanoscale, 2011, 3, 2461–2464.

Nanoscale, 2011, 3, 4150–4161 | 4161