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Multifunctional superparamagnetic MnO@SiO2 core/shell nanoparticles andtheir application for optical and magnetic resonance imaging†

Thomas D. Schladt,a Kerstin Koll,a Steve Pr€ufer,bd Heiko Bauer,a Filipe Natalio,a Oliver Dumele,a

Renugan Raidoo,a Stefan Weber,c Uwe Wolfrum,e Laura M. Schreiber,c Markus. P. Radsak,d Hansj€org Schildb

and Wolfgang Tremel*a

Received 19th October 2011, Accepted 27th February 2012

DOI: 10.1039/c2jm15320c

Highly biocompatible multifunctional nanocomposites consisting of monodisperse manganese oxide

nanoparticles with luminescent silica shells were synthesized by a combination of w/o-microemulsion

techniques and common sol–gel procedures. The nanoparticles were characterized by TEM analysis,

powder XRD, SQUID magnetometry, FT-IR, UV/vis and fluorescence spectroscopy and dynamic

light scattering. Due to the presence of hydrophilic poly(ethylene glycol) (PEG) chains on the SiO2

surface, the nanocomposites are highly soluble and stable in various aqueous solutions, including

physiological saline, buffer solutions and human blood serum. The average number of surface amino

groups available for ligand binding on the particles was determined using a colorimetric assay with

fluorescein isothiocyanate (FITC). This quantification is crucial for the drug loading capacity of the

nanoparticles. SiO2 encapsulated MnO@SiO2 nanoparticles were less prone to Mn-leaching compared

to nanoparticles coated with a conventional bi-functional dopamine–PEG ligand. The presence of

a silica shell did not change the magnetic properties significantly, and therefore, the MnO@SiO2

nanocomposite particles showed a T1 contrast with relaxivity values comparable to those of PEGylated

MnO nanoparticles. The cytotoxicity of the MnO@SiO2–PEG/NH2 nanoparticles was evaluated using

primary cells of the innate immune system with bone marrow-derived polymorphonuclear neutrophils

(BM-PMNs) as import phagocytes in the first line of defence against microbial pathogens, and bone

marrow-derived dendritic cells (BMDCs), major regulators of the adaptive immunity. MnO@SiO2–

PEG/NH2 nanoparticles have an acceptable toxicity profile and do not interact with BMDCs as shown

by the lack of activation and uptake.

Introduction

The design of new multifunctional nanomaterials that combine

different physical and chemical properties has become a distinct

driving force in modern-day materials science. Especially the

integration of magnetic nanomaterials with molecular biology

and biomedicine has evolved into a new research area, which is

now widely called nano-biotechnology.1 So far, magnetic

aInstitut f€ur Anorganische Chemie und Analytische Chemie, JohannesGutenberg-Universit€at, Duesbergweg 10-14, D-55099 Mainz, Germany.E-mail: tremel@uni-mainz.debInstitut f€ur Immunologie, Johannes Gutenberg-Universit€at, ObereZahlbacher Straße 67, D-55131 Mainz, GermanycInstitut f€ur Medizinische Physik, Klinik und Poliklinik f€ur Diagnostischeund Interventionelle Radiologie, Universit€atsklinikum, Langenbeckstraße1, D-55131 Mainz, GermanydThird Department of Medicine, Johannes Gutenberg University MedicalCenter, Mainz, GermanyeDepartment of Cell and Matrix Biology, Institute of Zoology, JohannesGutenberg University of Mainz, M€ullerweg 6, D-55099 Mainz, Germany

† Electronic supplementary information (ESI) available. See DOI:10.1039/c2jm15320c

This journal is ª The Royal Society of Chemistry 2012

nanoparticles have been used in various biomedical applications

ranging from protein separation, targeted drug delivery,

magnetic hyperthermia, and magnetic resonance imaging

(MRI).2–6 MRI is one of the most powerful non-invasive diag-

nostic techniques, as it provides unsurpassed 3D soft tissue

detail, without using ionizing radiation, deep tissue penetration

and high resolution, and therefore, has been applied for the

diagnosis of various diseases. The method is based on the

relaxation of protons in an external static magnetic field after

transmission of an excitation pulse. This process occurs

according to two different mechanisms: either by spin–lattice

relaxation (with a relaxation time T1) or by spin–spin relaxation

(with relaxation times T2 and T2*, respectively). As in any other

imaging technique, contrast agents are used in MRI to shorten

the respective relaxation times, and thus enhance the visibility of

pathological aberrations.7

The utilization of magnetic nanoparticles for these purposes is

quite reasonable, given the fact that their size, size distribution,

and morphology are easily controllable, which enables a precise

tuning of their magnetic properties. Superparamagnetic iron

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oxide nanoparticles (SPIONs) (i.e. magnetite (Fe3O4) and

maghemite (g-Fe2O3)) are, by far, the most intensively studied

materials in this field, owing to their high saturation magneti-

zation and low coercivity. They accelerate the spin–spin relaxa-

tion and thus there is a decrease in the transverse signal intensity

by generating local magnetic field inhomogeneities; therefore,

they are used as negative (T2) contrast agents. In fact, some iron

oxide nanoparticle probes have been approved for clinical use by

the Food and Drug Administration (FDA) of the United

States.7,8

However, T2 (or MR negative) contrast agents lead to

a decrease in signal intensity, and thus to a darkening in the MR

image. Positive (or T1) MR contrast agents, on the other hand,

accelerate the recovery of the longitudinal magnetization, leading

to a brightening in the corresponding MR image. These agents

usually comprise stable paramagnetic gadolinium complexes,

such as Gd–DTPA or Gd–DOTA, since free Gd3+ ions exhibit

a considerable toxic potential (e.g. high doses can lead to kidney

damages). As a consequence, special chelating ligands are

necessary to efficiently shield the central ion. However, this

approach limits the number of water molecules that can enter the

inner sphere of the contrast agent, which, in turn, results in

relatively low relaxivity (r1) values (typically 3.0–5.0 s�1 mM�1).

On the other hand, higher r1 values have recently been reported

for Gd-based nanoparticles, like Gd2O3 (with r1 in the range of

8.0–9.9 s�1 mM�1),9,10 GdPO4 (r1 ¼ 13.9 s�1 mM�1),11 or GdF3 (r1¼ 8.8 s�1 mM�1);12 however, they lack a precise control over size

and morphology, which limits their possible application.13

Recently, superparamagnetic MnO nanoparticles have also

proven to be potential candidates as T1 contrast agents, although

their relaxivity values are lower (typically with r1 in the range

between 0.2 and 2.0 s�1 mM�1).14–19 In the case of MnO, the

magnetic behavior results from the presence of uncompensated

magnetic moments on the surface of the nanoparticles.20

Most synthetic approaches for size and shape controlled

magnetic nanoparticles are based on the decomposition of

metal–organic compounds at high temperatures and in the

presence of hydrophobic surfactant molecules.21–24 In those

cases, a narrow size distribution is achieved by precise adjust-

ment of the individual synthetic parameters. However, efficient

surface modification strategies are needed to enhance both

hydrophilicity and biocompatibility of the nanoparticles and to

prevent both agglomeration in the physiological environment

and non-specific uptake by the reticuloendothelial system

(RES).25 Most of these strategies employ poly(ethylene glycol)

(PEG) as a hydrophilic linker, since it guarantees not only pro-

longed blood half-life and circulation but also reduced opsoni-

zation by serum proteins.26–29 Besides ligand exchange with either

bi-functional16,30,31 or multifunctional polymers3,32,33 and micellar

incorporation with functional amphiphilic polymers,34,35 silica

encapsulation is one of the most often used methods for surface

functionalization of inorganic nanomaterials.36

Apart from excellent biocompatibility, the presence of a silica

shell around metal oxide nanoparticles offers many advantages,

such as chemical and physical protection from the surrounding

environment, stability in aqueous media and a vast and robust

platform for further modification. In fact, the well developed

surface chemistry for silica micro- and nanoparticles provides the

opportunity to specifically tune the particle properties for later

9254 | J. Mater. Chem., 2012, 22, 9253–9262

applications.8,37–39 Additionally, the possibility of incorporating

fluorescent dyes inside the SiO2 shells has led to a wide extension

of applicability, owing to reduced photobleaching, blocked

quenching effects and suppression of energy transfer through the

silica matrix.40–43

In recent years, there has been tremendous progress in the

development of new strategies for silica encapsulation of inor-

ganic nanoparticles.44–52 However, the most often applied

procedures for the coating of hydrophobic nanoparticles are

based on the polymerization of silane monomers (usually tet-

raethyl orthosilicate (TEOS)) inside the micelles of reverse (or

water-in-oil (w/o)) microemulsions.53–56 The micelles comprise of

basic aqueous droplets, which are stabilized by a nonionic

surfactant in a continuous non-polar phase. Their size can easily

be tuned by varying the individual synthetic parameters,

including the molar water/surfactant ratio. Since the hydrolysis

of silane monomer only occurs inside the micelles or at the

water–oil interface, the resulting silica coated nanoparticles are

very homogeneous in size and shape, which is favorable

compared to particles obtained by a St€ober-type approach.57

Combining both the w/o microemulsion technique and the

common sol–gel methods in a one-pot reaction is thereby

a relatively new technique58 which can be applied to various

hydrophobic nanoparticles to create multifunctional nano-

composites and involves the successive addition of different

functional silanes. Furthermore, incorporation of fluorescent

dyes such as Atto 465 permits their detection during in vitro

studies.59 We demonstrate that multifunctional silica coated

MnO nanoparticles are stable against aggregation in water,

physiological saline, and human blood serum over several weeks.

Besides colloidal stability, the quantification of the particle

bound target molecules is a matter of interest because the drug

loading capacity is decisive for the dose rate determination and

a successful application of the particles in drug delivery. Quan-

tification of the particle bound molecules is an analytical chal-

lenge, because either the lack or the superposition of

chromophores excludes direct spectrophotometrical detection by

excitation of fluorescence. We have determined the average

number of surface amino groups available for ligand binding on

each particle using a colorimetric assay with fluorescein iso-

thiocyanate (FITC).

Furthermore, we show that the silica shell has only a negligible

influence on the magnetic properties, and that therefore, the

MnO@SiO2 nanoparticles still exhibit a considerable T1 MR

contrast enhancement effect. We have designed previously

pathogen-mimicking metal oxide nanoparticles with the ability

to enter cells and to selectively target and activate the TLR9

pathway.32,60 Here, nanoparticle surface modification provides

an efficient and convenient means for loading immunostimula-

tory oligonucleotides. Therefore, the cytotoxic behavior of the

silica encapsulated MnO nanoparticles was studied for two

primary immune cells: dendritic cells (DC) and poly-

morphonuclear neutrophils (PMNs). DCs belong to the group of

antigen-presenting cells (APCs) and are able to initiate and

modulate the adaptive immune response. PMNs are the most

abundant type of leukocytes in the human blood. As part of the

innate immune system they play a central role in the defence

against invading microbial pathogens. The MnO@SiO2 nano-

particles showed negligible cytotoxicity for both cell types.

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Additionally, we analysed the activation and uptake of BMDC

after incubation with MnO@SiO2 nanoparticles and we see

neither activation nor unspecific uptake of the nanoparticles. So

we can conclude that the described MnO@SiO2 nanoparticles

are suitable candidates for in vivo applications.

Experimental section

Synthesis

Materials. Manganese chloride tetrahydrate (MnCl2$4H2O)

(99.9%), oleic acid (90%), sodium hydroxide (99%), 1-octadecene

(90%), Igepal CO-520, ammonium hydroxide (25%, aqueous

solution), tetraethoxysilane (TEOS) (99.999%), 3-amino-

propyltriethoxysilane (APS) (>99%), fluorescein isothiocyanate

(FITC), and DMF (>99.9%, extra dry) were purchased from

Aldrich. Atto 465-NHS-ester was from Fluka, and 2-methoxy

(polyethyleneoxy)propyltrimethoxysilane (PEG–silane, n ¼ 6–9)

(90%) was from ABCR. Methanol, hexane, cyclohexane, and

acetone were of analytical grade (Fisher Scientific). All chemicals

were used without further purification.

Synthesis of MnO nanoparticles. Monodisperse MnO nano-

particles were prepared by the decomposition of manganese

oleate in high boiling point solvents as described previously.20,61

Briefly, 2 mmol of manganese oleate were mixed with 10 g of 1-

octadecene, degassed at 70 �C and slowly heated to reflux with

a defined temperature program. The particles were washed by

repeated precipitation with acetone, centrifugation and disper-

sion in hexane.

Synthesis of APS–Atto 465. APS–Atto 465 was prepared by

reacting 1 mg (�2 mmol) of Atto 465-NHS-ester with 23.5 mL

(100 mmol) of APS in 500 mL of dry DMF for 4 hours in a shaker

at 20 �C in the dark. The solution was kept as a stock solution at

�20 �C in the dark.

Silica encapsulation of MnO nanoparticles. Silica encapsula-

tion of monodisperse nanoparticles was performed as described

previously, slightly adjusting the method.58,59 The hydrophobic

MnO nanoparticles were encapsulated with SiO2 by a w/o

microemulsion technique using cyclohexane as the oil phase,

aqueous NH4OH solution as the water phase and Igepal CO-520

as non-ionic surfactant. For the encapsulation process, Igepal

CO-520 (2.0 g) was dissolved in 35 mL of cyclohexane and

degassed by bubbling a gentle stream of argon through the

solution for 30 minutes. The nanoparticles were added to the

solution and the degassing procedure was continued for another

15 minutes. Subsequently, aqueous NH4OH (150 mL) was added

to initiate the micelle formation. This process could be observed

as a temporary cloudiness in the solution. After 5 minutes TEOS

(112 mL, 0.5 mmol) and APS–Atto 465 were added and the

reaction mixture was stirred under argon protection overnight.

For further functionalization of the silica shell, PEG–silane was

added to the solution. The covalent attachment of PEG on the

surface led to complete precipitation of the MnO@SiO2 nano-

particles after 2 h. The nanoparticles were collected by centri-

fugation (5000 rpm for 5 min) and washed thoroughly by

repeated dispersion in acetone and precipitation with hexane.

This journal is ª The Royal Society of Chemistry 2012

The fact that the resulting particles were easily soluble in polar

solvents, such as acetone or ethanol, is a clear indication of the

successful hydrophilic modification of the initially hydrophobic

MnO surface. After complete removal of any unreacted silanes

and surfactant molecules, the MnO@SiO2–PEG nanoparticles

were dissolved in 20 mL of acetone. Amino groups were intro-

duced by addition of APS (150 mL, 0.64 mmol) and 150 mL

aqueous NH4OH. The reaction was continued for another 2 h

followed by concentration of the solution to 2 mL and precipi-

tation of the MnO@SiO2PEG/NH2 nanoparticles with 2 mL of

hexane. Again, the resulting particles were thoroughly washed by

repeated dissolving and precipitating with acetone and hexane

accompanied by centrifugation (5000 rpm for 5 min). The

MnO@SiO2–PEG/NH2 nanoparticles obtained by this method

were very easily soluble and extremely stable in various aqueous

media, including deionized water, physiological saline, and

different buffer solutions.

Nanoparticle characterization

The particles were characterized by means of powder X-ray

diffraction (XRD), transmission electron microscopy (TEM),

Fourier transformed infrared spectroscopy (FT-IR), atomic

absorption spectroscopy (AAS), UV-vis and fluorescence spec-

troscopy, and dynamic light scattering (DLS). The magnetic

properties of the MnO@SiO2 nanoparticles were investigated

with a superconducting quantum interference device (SQUID,

Quantum Design MPMS-XL). XRD measurements were per-

formed on a Bruker D8 Advance diffractometer equipped with

a Sol-X energy-dispersive detector and operating with Mo Ka

radiation. Samples for transmission electron microscopy were

prepared by placing a drop of dilute nanoparticle solution in the

appropriate solvent (hexane, acetone, or water) on a carbon

coated copper grid. Low-resolution TEM images and ED

patterns were recorded on a Philips EM420 microscope oper-

ating at an acceleration voltage of 120 kV. FT-IR spectra were

acquired on a Mattson Instruments 2030 Galaxy-FT-IR spec-

trometer. For AAS measurements (Perkin-Elmer 5100 ZL),

aliquots of aqueous nanoparticle solutions were treated with

conc. HNO3 at 90�C for 30 minutes followed by adjustment of

the pH-value to 3 with NH4OH. UV-vis spectroscopy was

carried out on a Perkin-Elmer Lambda 19 UV-vis/NIR spec-

trometer. Fluorescence spectra were acquired on a Spex Fluo-

rolog 1680/81. Determinations of the zeta-potential and dynamic

light scattering were performed on aMalvern Zetasizer Nano ZS.

Mn leaching experiments

To evaluate the stability of the MnO nanoparticles encapsulated

by SiO2 in comparison to MnO nanoparticles functionalized by

a bi-functional dopamine–PEG ligand, aqueous solutions of

both kinds of particles were dialyzed, in a cellulose membrane

with molecular weight cutoff (MWCO) of 3500 (Spectra/Por 3,

SpectrumLabs), against deionized water. The variation in the

Mn concentration over a period of four weeks was monitored by

atomic absorption spectroscopy (AAS) on aliquots taken after

definite time intervals. In a typical experiment, 10 mg of the

concerning functionalized nanoparticles were dissolved in 5 mL

of deionized water, loaded into the dialysis tube, and placed in

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a 2000 mL glass cylinder filled with deionized water. The cylinder

was sealed to prevent any contamination. The dialysis was

carried out for four weeks with the surrounding water being

replaced every second day. Aliquots of 50 mL were carefully

extracted after fixed time intervals.

Quantification of surface amino groups

The NH2-determination scheme is presented in Fig. S1 (ESI†).

To determine the average number of amino groups on each

particle, the MnO@SiO2–PEG/NH2 nanoparticles were treated

with fluorescein isothiocyanate (FITC). After complete reaction,

the amount of unreacted FITC in the supernatant was deter-

mined by UV-vis spectroscopy. In a typical experiment the

prepared MnO@SiO2–PEG/NH2 particles were dissolved in

10 mL of acetone. An aliquot of 1 mL of this stock solution was

treated with an equal amount of hexane to completely precipitate

the nanoparticles followed by centrifugation (5 min, 9000 rpm).

Subsequently, the particles were resuspended and diluted in

acetone by a factor of 1 : 100, before FITC was added according

to the maximum possible amount of APS in the dilute sample.

After the reaction was completed (one hour) the particles were

precipitated by adding exactly 1 mL of hexane followed by

centrifugation (10 min, 13 300 rpm). The concentration of

unreacted FITC in the supernatant was determined using a cali-

bration curve according to the Lambert–Beer law. The difference

in the amount of FITC before and after the reaction was used to

estimate the amount of free amino groups in the sample. With

this result, the average number of free amino groups on each

particle can be calculated by correlating the total amount of NH2

groups with the average number of nanoparticles in the sample.

Scheme 1 Surface modification procedure to produce multi-functional

silica coated MnO nanoparticles by successive addition of silica

precursors.

Cell culture

Generation and culture of bone marrow-derived DC (BMDC).

BMDCs were generated from C57BL/6 mice as described

previously.62 Briefly, bone marrow from 6–8 week old mice was

isolated and cultured in medium (Iscove’s medium supplemented

with 5% fetal calve serum (FCS), 1% glutamate, 1% sodium

pyruvate) and GM-CSF (50 ng mL�1) at 37 �C with 5% CO2.

These cells were typically >85% CD11c+H2-Ab+ and showed the

typical BMDC surface marker profile (CD80low and CD86low) as

determined by flow cytometry measurements. The medium was

changed on days 3 and 5. Cells were used for the experiments at

day 7 as immature BMDCs.

Purification and culture of bone marrow-derived poly-

morphonuclear neutrophils (BM-PMNs). BM-PMNs were puri-

fied by magnetic beads (MACS from Miltenyi, Bergisch

Gladbach, Germany) using Ly6G/C specific antibodies (clone

Gr-1) according to the manufacturer’s protocol. BM-PMNs were

cultured in medium (Iscove’s medium supplemented with 5%

fetal calve serum (FCS), 1% glutamate, 1% sodium pyruvate) at

37 �C with 5% CO2. Cell purity (Ly6-GhiCD11bhi) was generally

greater than 90% as assessed by flow cytometry measurements.

Determination of apoptosis, cytokine release and uptake of the

nanoparticles. BMDCs or BM-PMNs (2 � 105 cells per well) were

cultured in 96-well plates (Greiner) with titrated concentrations of

9256 | J. Mater. Chem., 2012, 22, 9253–9262

MnO@SiO2–PEG/NH2 nanoparticles, cyclo-heximide (10mgmL�1,

Fluka), the TLR4 agonist LPS (100 ng mL�1, Salmonella typhimu-

rium, Sigma, Deisenhofen, Germany) or left untreated and kept at

37 �C with 5% CO2 until analysis. For calcein labelling, cells were

incubated for 30 minutes at 37 �C and 5% CO2 with 0.5 mg mL�1

calcein (Invitrogen, Heidelberg, Germany) in medium, then washed

twice.

For the detection of apoptosis, BM-PMNs were harvested

after 24 h (because PMNs are short-lived cells) or BMDCs after

72 h, respectively. Afterwards the cells were analyzed for

apoptosis using either Annexin-V (BD Pharmingen)/propidium

iodide (PI, Fluka) staining or Nicoletti staining for fragmented

nuclear DNA using previously described protocols.63

The release of IL-6 and IL-12 by BMDC into culture super-

natants was analyzed by standard enzyme-linked immunosor-

bent assay (ELISA) described elsewhere.64

The uptake of the MnO@SiO2–PEG/NH2 nanoparticles was

revealed by FACS analysis and electron microscopy. Electron

microscopy was performed using a Tecnai 12 transmission elec-

tron microscope (FEI Company). BMDCs (4� 106 cells per well)

were cultured in 6-well plates (Greiner) with 30 mg mL�1 of

MnO@SiO2–PEG/NH2 nanoparticles. After 24 h the BMDCs

were prepared as described previously.65

Magnetic resonance imaging

MR signal enhancement effects of MnO@SiO2 nanocomposite

particles were measured with different Mn concentrations on

a clinical 3.0 T MRI scanner (Magnetom Trio, Siemens Medical

Solutions, Erlangen, Germany). Signal reception and radio

frequency (RF) excitation was performed using an 8-channel

knee coil. For the T1-measurement, a saturation prepared (SR)

snapshot fast low angle shot (SR-TurboFLASH) pulse sequence

with repetition time (TR)/echo time/flip angle ¼ 3.0 ms/1.5 ms/

20� was used with varying saturation times starting from 20 ms

up to 8000 ms. For measuring the T2 relaxation time, a multi-

echo spin-echo pulse sequence with a total of 32 echos and

TR ¼ 5000 ms was used; the echo time was varied from 7 ms to

224 ms. In a second T2 measurement TE was varied from 15 ms

up to 480 ms.

Results and discussion

MnO nanoparticles: synthesis and properties

Particle synthesis and silica coating. The synthesis scheme for

the preparation of the multifunctional silica coated MnO

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nanocomposites is illustrated in Scheme 1. First, hydrophobic

oleate capped MnO nanoparticles are synthesized according to

a previously published method.20,61 In the second step, the

nanoparticles are coated with a thin shell of SiO2 by hydrolysis of

tetraethylorthosilicate (TEOS) in a reverse microemulsion. The

shell thickness is easily adjustable by variation of the synthetic

parameters, including micelle size, amount of TEOS, or reaction

time. During this procedure, a fluorescent dye (Atto 465) is

additionally incorporated into the silica shell, to allow optical

detection of the nanoparticles by fluorescence microscopy. In the

following steps, further functionalization is achieved by

condensation of a PEG–silane conjugate (PEG–TES) and 3-

aminopropyltriethoxysilane (APS) onto the nanocomposite

surface. The attachment of PEG groups not only increases the

hydrophilic character, but also ensures biocompatibility of the

magnetic nanoparticles in physiological environment, whereas

the presence of free amino groups on the particle surface enables

further conjugation. Consequently, the MnO@SiO2–PEG/NH2

nanocomposite particles are readily soluble in polar solvents,

such as ethanol or acetone, which permits a simple purification

procedure by repeated precipitation/centrifugation/resuspension

with hexane and ethanol/acetone.

Nanoparticle characterization. The successive silane-function-

alization steps of the MnO nanoparticles were monitored by

transmission electron microscopy (TEM) and Fourier-trans-

formed infrared (FT-IR) spectroscopy. Representative TEM

images of the different synthesis stages are displayed in Fig. 1.

Fig. 1a shows as-prepared oleate capped MnO nanoparticles

before functionalization with SiO2. The particles are uniform in

Fig. 1 TEM images ‘‘naked’’ and silica functionalized MnO nano-

particles. (a) As synthesizedMnO nanoparticles with an average diameter

of nm. (b) MnO nanoparticles coated with a uniform shell of silica after

hydrolysis of TEOS. (c) MnO@SiO2 nanoparticles after conjugation with

PEG–silane and APS, no significant aggregation is visible. (d) Enlarged

view of MnO@SiO2–PEG/NH2 nanoparticles in image (c).

This journal is ª The Royal Society of Chemistry 2012

size and shape with an average diameter of 10 nm (s# 5%). After

the first modification step the particles are covered by a 3 nm

thick silica shell (Fig. 1b) and retain their narrow size distribu-

tion. Due to the higher electron density, MnO appears darker in

the TEM images compared to SiO2. It should be noted, that no

multiple MnO nanoparticles per SiO2 shell are observed. This

can be ascribed to an ideal micelle size during the reverse

microemulsion step, in which each micelle contains only one

MnO nanoparticle. Fig. 1c and d show the final MnO@SiO2–

PEG/NH2 nanoparticles after complete functionalization. There

is a slight increase in shell thickness to 4–5 nm, however, no

significant particle aggregation is observable and the nano-

particles appear uniform and well separated. The virtually

identical values for the nanoparticle sizes by TEM and DLS

demonstrate their excellent colloidal stability.

The presence of the individual functionalities on the MnO

nanoparticle surface was confirmed by FT-IR spectroscopy.

Fig. 2a displays FT-IR spectra of the MnO@SiO2 nano-

composites after each functionalization step (top to bottom). The

spectrum of as-prepared oleate capped MnO nanoparticles

(black line, top) exhibits characteristic vibrational bands at 1555

and 1410 cm�1 which can be assigned to the asymmetric and

symmetric stretching modes of the carboxylate group of the

oleate molecules.25 The weak absorption band at 2956 cm�1

results from the asymmetric CH3-stretching mode. Two bands at

2922 and 2852 cm�1 can be assigned to the symmetric and

asymmetric CH2- stretching modes of the alkyl chain, respec-

tively. After the first step (MnO@SiO2, red line), the carboxylate

bands have almost disappeared completely, however, strong and

broad absorption bands appear in the spectral region between

1155 and 1020 cm�1, that can be associated with asymmetric O–

Si–O stretching vibrations. This is a clear indication of the

successful exchange of the oleate layer by SiO2. The coupling of

PEG molecules on the surface of the MnO@SiO2 nano-

composites (MnO@SiO2–PEG, blue line) is accompanied by the

appearance of two new bands at 1463 and 1377 cm�1. They are

due to the symmetric stretching vibrations of the C–O–C ether

groups inside the PEG moieties. Additionally, the asymmetric

and symmetric CH3- and CH2- stretching vibrations at 2954,

2918, and 2869 cm�1 appear more pronounced, owing to the

PEG alkyl structure. Finally, the presence of free amino groups

Fig. 2 (a) FT-IR spectra of MnO nanoparticles recorded after each

functionalization step during the synthesis (from top to bottom). (b)

Powder XRD pattern of ‘‘naked’’ and silica coated MnO nanoparticles.

The red bars indicate the expected reflection positions.

J. Mater. Chem., 2012, 22, 9253–9262 | 9257

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on MnO@SiO2–PEG/NH2 (green line) was confirmed by the

occurrence of a band at 1636 cm�1 which can be assigned to the

H–N–H scissoring vibration, and an absorption band at

800 cm�1, that is due to the NH2- wagging vibration. Unfortu-

nately, the expected asymmetric and symmetric NH2- stretching

vibrations between 3300 and 3400 cm�1 are not visible among the

broad OH- oscillation in the concerned spectral region. More-

over, the CN- stretching vibration, which is expected around

1080 cm�1, is superimposed by the strong absorption of SiO2.

Phase purity of the silica-coated MnO nanoparticles was

investigated by powder X-ray diffraction. For comparison, the

powder patterns of pure MnO nanoparticles and MnO@SiO2

NPs are displayed in Fig. 2b. In both cases the observed inten-

sities match well with the cubic rock salt structure of MnO (cF8,

Fm�3m). Therefore, it has to be estimated that no significant

oxidation to Mn3O4 has occurred during the synthesis. However,

the presence of the silica shell around the nanoparticles leads to

a considerable increase in background intensity which impedes

a definite assignment.

Magnetic properties. As mentioned before, the magnetic

behavior of MnO nanoparticles results from the presence of

uncompensated spins on the particle surface. Hence, it was

expected, that the silica shell has a considerable influence on the

magnetic behavior of the nanoparticles. The magnetic properties

of ‘‘naked’’ and silica coated MnO nanoparticles with shell

thicknesses of 2 and 5 nm were studied using a superconducting

quantum interference device (SQUID) (see Fig. 3). Field-

dependent magnetization measurement revealed super-

paramagnetic behavior at 300 K and weak ferromagnetic

behavior at 5 K. Interestingly, the effect of the silica coating is by

far less pronounced as expected from literature reports on other

silica coated magnetic nanoparticles, such as magnetite.62 In

contrast to Tan et al. we observed only a slight decrease in

magnetization at 5 T with increasing silica shell thickness.

Temperature-dependent magnetization curves of the corre-

sponding samples (see insets in Fig. 3) further confirm the

observed superparamagnetism. The magnetic blocking

temperature TB, which is located at the maximum of the zero-

field-cooled curves, is a measure of the transition from the

superparamagnetic to the ferromagnetic state. It indicates the

temperature below which the thermal energy is insufficient to

freely flip the magnetic moments of the nanoparticles, therefore,

inducing ferromagnetism. For bare MnO nanoparticles TB was

Fig. 3 Hysteresis loops recorded at 300 and 5 K of (a) ‘‘naked’’ MnO

nanoparticles, (b) MnO@SiO2 nanoparticles with a silica shell thickness

of 2 nm, and (c) MnO@SiO2 nanoparticles with a silica shell thickness of

5 nm. The insets display the corresponding zero-field-cooled field-cooled

(ZFC-FC) curves recorded with an applied induction field of 100 Oe.

9258 | J. Mater. Chem., 2012, 22, 9253–9262

reported to be in the range between 10 and 20 K.20,66,67 Indeed,

the observed TB of 17 K for the uncoated MnO nanoparticles

(inset Fig. 3) is in good agreement with the literature values.

Upon silica encapsulation, the magnetic blocking temperature

increases to 23 K for the sample with a 2 nm SiO2 shell, and to

24 K for the sample containing a 5 nm SiO2 shell, respectively.

This circumstance can be explained by considering the larger

effective mass of the MnO@SiO2 nanoparticles compared to

pure MnO nanoparticles. A larger mass leads to a higher thermal

energy which is necessary to randomly flip the magnetic

moments. A similar behavior has recently been reported for

Au@MnO nanoparticles.33

UV/vis and fluorescence spectroscopy. As mentioned above, the

incorporation of fluorescent dyes into a silica matrix offers many

advantages compared to their attachment on the outside of the

nanocomposites. However, a sufficient integration of free dyes

into SiO2 can only be achieved after prior modification of the dye

molecules. For this purpose, the NHS-ester of the fluorescent dye

Atto 465 was reacted with APS in anhydrous DMF, to form

a reactive silane–dye conjugate. After that, TEOS and the APS–

Atto 465 conjugate were injected into the reverse microemulsion

at the same time. Since the hydrolysis of both components

occurred simultaneously inside the micelles, the dye was effi-

ciently incorporated into the silica shell, as confirmed by UV-vis

and fluorescence spectroscopy (Fig. 4). The UV-vis absorption

spectra of pure MnO@SiO2, pure Atto 465, and Atto 465 doped

MnO@SiO2 (MnO@SiO2:A465) are displayed in Fig. 4a. Pure

MnO@SiO2 nanocomposites containing no dye (black line)

exhibit no prominent absorption characteristics, the absorbance

increases gradually with decreasing wavelength. On the other

hand, Atto 465-doped MnO@SiO2 nanoparticles (blue line)

show a maximum in absorbance at 470 nm, even if the dye

concentration is considerably low (molar ratio Atto 465/

TEOS ¼ 0.04%). However, the free dye (green line) exhibits

a sharp maximum at 460 nm, and therefore, it has to be assumed,

that the presence of the silica matrix around the dye molecules

leads to a red-shift in the absorbance of approximately 10 nm.

This observation is in accordance with previous reports on silica

nanoparticles containing fluorescent dyes.40,41 Furthermore, the

fluorescence properties of the nanocomposites (Fig. 4b) revealed

Fig. 4 (a) UV-vis-spectra of pureMnO@SiO2 nanoparticles (black line),

pure Atto 465 dye (green line), and MnO@SiO2:Atto 465 nanoparticles

(blue line) in water. (b) Photoluminescence spectra of pure Atto 465 dye

(green line) and MnO@SiO2:Atto 465 nanoparticles (blue line) at room

temperature in water (inset: fluorescence microscopy image of MnO@-

SiO2:A465 nanoparticles, scale bar 0.5 mm).

This journal is ª The Royal Society of Chemistry 2012

Fig. 5 T1 weighted images of physiological saline solutions containing

MnO@SiO2–PEG/NH2 nanoparticles in different concentrations. The

yellow circle indicates an air bubble in the sample tube, which occurred

during sample placement into the MRI scanner.

Fig. 6 (a) T1 (right) and T2 (left) relaxation rates as a function of

manganese concentration. (b) Evolution of the manganese content during

the leaching experiment for silica coated and dopamine–PEG function-

alized MnO nanoparticles.

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a similar behavior. Free Atto 465 fluoresces with a maximum

emission intensity at 508 nm (green line), whereas for MnO@-

SiO2:A465 (blue line), the maximum is shifted to 512 nm. The

inset in Fig. 4b shows MnO@SiO2:A465 nanoparticles observed

under a fluorescent microscope. The particles are clearly visible

as green dots and appear well separated. The different intensities

observed in this figure inset can be explained in the following

manner: (i) the optical resolution of the fluorescence microscope

does not allow us to distinguish single nanoparticles and thus

their proximity (no clustering) leads to the formation of

a broader emission and (ii) nanoparticles were observed in

aqueous solutions (e.g. aqueous droplet) and consequently their

distribution occurs in three-dimensional space. As a result, their

proximity in all directions can generate a more intense emission.

These results substantiate the potential to utilize these nano-

particles as fluorescent probes for optical imaging.

Colloidal stability and quantification of NH2 groups. A key

requirement for later biomedical application of different nano-

materials is, besides a low toxicity, a sufficient stability in phys-

iological environments. For this purpose, we investigated the

colloidal stability of our silica encapsulated MnO nanoparticles

in various aqueous solutions, including deionized water, physi-

ological saline and human blood serum. Fig. S3 (ESI†) shows

photographs of the different nanoparticle solutions in water and

human blood serum, respectively, after 24 h at 22 �C. Zeta-potential (x) measurements, conducted on pure MnO@SiO2,

MnO@SiO2–PEG and MnO@SiO2–PEG/NH2 nanoparticles,

revealed, that the net surface charges are altered upon additional

surface functionalization. For MnO nanoparticles coated with

bare silica, the determined surface charge is x ¼ �40.0 mV,

whereas for MnO@SiO2–PEG the value is �45.0 mV, both of

which are due to the presence of deprotonated silanol groups

(–Si–O�, pKa ¼ 7.0). Since, amino groups (pKa ¼ 9.0) usually

occur as protonated ammonium moieties (–NH3+) at ambient

pH, the introduction of APS onto the silica surface should be

accompanied by shift of the zeta-potential into the positive

regime.68 Indeed, the expected alteration was observed with

x ¼ �24.4 mV. Furthermore, dynamic light scattering (DLS)

measurements confirmed the excellent colloidal stability of the

nanoparticles (see Fig. S4, ESI†). The determination of surface

bound amino groups by the above mentioned colorimetric

method (see Experimental section) revealed an average NH2-

group concentration of 2.4 mmol per mg nanoparticles. This

corresponds approximately to an average number of 9.3 million

NH2 groups on the surface of a single particle. The calculation

method is described in the ESI (Fig. S1†).

MR imaging. To investigate the MR signal enhancement

effects, aqueous solutions of silica functionalized MnO nano-

particles, at different Mn concentrations, were measured on

a clinical 3.0 T MRI scanner. As mentioned above, MnO

nanoparticles are known to shorten the longitudinal relaxation

time (T1), which leads to a brightening in the corresponding MR

image, compared to samples without a contrast agent. Prior to

the experiment, MnO@SiO2–PEG/NH2 nanoparticles were

diluted in physiological saline solution yielding concentrations of

0 to 5.3 mM (with respect to manganese, determined by AAS).

Fig. 5 displays T1-weighted phantom MR images of these

This journal is ª The Royal Society of Chemistry 2012

solutions. It is clearly evident that the brightness of the image

increases with increasing manganese (and therefore, MnO@-

SiO2–PEG/NH2 nanoparticle) concentration. For an exact

quantification of the MR signal enhancing properties, the

relaxivities r1 and r2 were determined according to the general

relation: 1/T1 ¼ 1/T10 + r1c and 1/T2 ¼ 1/T2

0 + r2c, in which T1

and T2 are the measured longitudinal and transverse relaxation

times, respectively, T10 and T2

0 are the corresponding relaxation

times in the pure media, and c is the concentration of the contrast

agent. Fig. 6a shows plots of 1/T1 and 1/T2 versus the Mn

concentration, in which a linear increase is obvious in both cases.

The r1 and r2 values, derived from the slope of the curve, are 0.47

and 2.85 mM�1 s�1, respectively, which is in good agreement with

earlier reports on manganese oxide nanoparticles.14,15,56 Addi-

tionally, the relaxivity ratio (r2/r1 ¼ 6.06) is considerably low,

which indeed suggests that the silica encapsulated MnO nano-

particles are potential candidates as T1 contrast agents.

Leaching experiments. Manganese leaching experiments were

carried out to evaluate the liability of the manganese oxide cores

inside the silica shells for degradation in aqueous solution. For

this purpose, solutions of MnO@SiO2–PEG nanoparticles in

water were transferred into dialysis membranes and dialyzed

against deionized water (18 MU). It should be noted, that, in

contrast to the stability tests (see above), no particle aggregation

was observed for the MnO@SiO2–PEG/NH2 particles during the

experiment, which, most likely, is due to the absence of surface

amino groups, and therefore, a lack of attractive ionic interaction

between the particles. The manganese content inside the

J. Mater. Chem., 2012, 22, 9253–9262 | 9259

Fig. 7 BM-PMNs (a and b) or BMDCs (c and d) (2 � 105 per well) were

incubated in the presence of the indicated concentration of MnO@SiO2–

PEG/NH2 nanoparticles or left untreated (neg.) and assayed for the

induction of apoptosis. Cycloheximide (10 mg mL�1, CHX) as a strong

inducer of apoptosis served as positive control, while the TLR4 agonist

LPS (100 ng mL�1), that is well known to delay the constitutive apoptosis

especially in PMN, was used as an additional control (a and b).63 BM-

PMNs were harvested after 24 h of in vitro culture with the indicated

stimuli and assayed for apoptosis by staining with Annexin-V/PI (a) or by

Nicoletti staining (b). (c and d) BMDCs were harvested after 72 h of

culture with the different stimuli and analysed for apoptosis by staining

with Annexin-V/PI (c) or by Nicoletti staining (d). The depicted data

show the cumulative results of two independent experiments each assayed

in duplicate wells (mean +/� SD percentage of cell viability of one

representative experiment assayed in duplicate wells). (*) indicates

a significant difference (p < 0.05) compared to the untreated control by

Student’s t test.

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membranes was monitored by AAS measurements over a period

of four weeks. For comparison, an aqueous solution of manga-

nese oxide nanoparticles, which were stabilized by a bi-functional

dopamine–PEG ligand (DA–PEG), rather than a robust silica

shell, was treated in the same way. The results, presented in

Fig. 6b, show an obvious trend: without the protection of a SiO2

shell, the Mn content decreased to almost 75% within 28 days,

whereas only a minor depletion of �1.5% was observed in the

MnO@SiO2–PEG sample, over the same time period. These

results elucidate the advantage of silica encapsulation of MnO

nanoparticles for potential biomedical applications. Addition-

ally, TEM measurements confirmed the expected degradation of

the DA–PEG functionalized nanoparticles. TEM images of both

samples after the leaching experiments are shown in Fig. S2

(ESI†). The SiO2 modified nanoparticles retained almost their

initial size and shape. The DA–PEG–MnO nanoparticles, on the

other hand, although not being dramatically reduced in size,

appear rougher and less homogeneous, which is a clear indica-

tion that dissolution has taken place on the particle surface.

Cytotoxicity. The effect of the MnO@SiO2–PEG/NH2 nano-

particles was evaluated using primary cells of the innate immune

system. For this purpose we used two major phagocytic cell

types: (i) bone marrow-derived polymorphonuclear neutrophils

(BM-PMNs) as import phagocytes in the first line of defence

against microbial pathogens and (ii) bone marrow-derived DC

(BMDC), major regulators of the adaptive immunity. For the

assessment of apoptosis two independent assays were used: (i)

Annexin-V/PI staining identifies early apoptotic events through

detection of externalized phosphatidylserine on the cell

membrane and (ii) Nicoletti staining detects fragmented DNA as

a later apoptotic event.63 Firstly, we examined the induction of

apoptosis in BM-PMN in the presence of titrated concentrations

of MnO@SiO2–PEG/NH2 nanoparticles as these cells are known

to be highly sensitive to external (i.e. bacterial) stimuli and

undergo apoptosis rapidly in vitro unless adequately activated.62

As depicted in Fig. 7, after 24 h we observed no differences

between the short lived BM-PMN treated with titrated concen-

trations of MnO@SiO2–PEG/NH2 nanoparticles and non-stim-

ulated cells (Fig. 7a and b) indicating that these nanoparticles do

not have any significant toxic effects on this cell type. Secondly,

we evaluated the BMDC apoptosis rate in the presence of

MnO@SiO2–PEG/NH2 nanoparticles as these cells are known to

be master regulators for the initiation of adaptive immune

responses.69 They act as resting sentinels in the periphery until

they get activated through a variety of microbial products as well

as tissue damage. For BMDC, a decrease in cell viability was

only observed at the highest concentration of MnO@SiO2–PEG/

NH2 nanoparticles (30 mg mL�1) after 72 h suggesting that these

nanoparticles exert toxic effects only at high concentrations on

primary cells. The higher cell viability in the Nicoletti staining

(Fig. 7b and d) compared with the corresponding Annexin-V/PI

staining (Fig. 7a and c) is in agreement with the fact that the

Nicoletti staining detects late apoptotic events whereas the

Annexin-V/PI staining reveals early signs of apoptosis.

BMDC activation and nanoparticle uptake. BMDCs initiate

and modulate the adaptive immune responses. For this purpose

they need to be in an activated state, indicated i.e. by high levels

9260 | J. Mater. Chem., 2012, 22, 9253–9262

of co-stimulatory molecules such as CD40, CD70, CD80 or

CD86 and the secretion of inflammatory cytokines like IL-6 and

IL-12. Therefore, levels of IL-6 and IL-12 were analysed post

incubation with titrated concentrations of MnO@SiO2–PEG/

NH2 nanoparticles with the standard ELISA technique (Fig. 8a).

We detected no significant differences between unstimulated and

even high dose MnO@SiO2–PEG/NH2 nanoparticles treated

BMDC. Only the TLR4 agonist LPS induced the secretion of

high amounts of IL-6 and IL-12. So we conclude that the

MnO@SiO2–PEG/NH2 nanoparticles do not activate the

BMDCs. For ingestion experiments, BMDCs were incubated

with 30 mg mL�1 of MnO@SiO2–PEG/NH2 nanoparticles and

after 24 h the specific uptake was quantified with FACS analysis

This journal is ª The Royal Society of Chemistry 2012

Fig. 8 (aand c)BMDCs(2� 105 perwell)were incubated in thepresenceof

the indicated concentration ofMnO@SiO2–PEG/NH2 nanoparticles or left

untreated (neg). As positive control the TLR4 agonist LPS (100 ng mL�1)

and Calcein (Cal., 0.5 mg mL�1) were used. (a) After 48 h of stimulation,

culture supernatants were collected for ELISA analysis of IL-6 (open bar)

and IL-12 (black bar). All data are presented asmean and standard error of

mean of duplicate wells from one representative of at least two independent

experiments. (*) indicates a significant difference (p< 0.05) compared to the

untreated control by Student’s t test. (b) Transmission electron microscopy

analysis of BMDCthat were incubatedwith 30 mgmL�1MnO@SiO2–PEG/

NH2 nanoparticles After 24 h BMDC were processed for conventional

electron microscopy. (c) The uptake of MnO@SiO2–PEG/NH2 nano-

particles was revealed by FACS analysis after 24 h. Shown is the mean

fluorescence intensity (MFI).

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and transmission electron microscopy. We were unable to detect

MnO@SiO2–PEG/NH2 nanoparticles inside the BMDC or their

accumulation at the plasmamembrane (Fig. 8b). In line with this,

we did not observe any uptake of fluorescent MnO@SiO2–PEG/

NH2 nanoparticles by FACS at any time point (Fig. 8c), by

confocal fluorescence microscopy (data not shown) or by MRI

(data not shown). These data suggest that the MnO@SiO2–PEG/

NH2 nanoparticles are not taken up by BMDC. Taken together,

the results obtained indicate that the MnO@SiO2–PEG/NH2

nanoparticles can be expected to have an acceptable toxicity

profile and BMDCs show no uptake of MnO@SiO2–PEG/NH2

nanoparticles or any activated phenotype. Hence, MnO@SiO2–

PEG/NH2 nanoparticles appear suitable for in vivo applications.

Conclusions

In summary, we have demonstrated a highly reproducible way to

prepare biocompatible multifunctional MnO@SiO2

This journal is ª The Royal Society of Chemistry 2012

nanocomposites. The procedure combines a water-in-oil micro-

emulsion, to create an initial silica shell around MnO, and

common St€ober-like processes to condense further functional-

ities, such as PEG or NH2-groups, on the silica surface. The silica

encapsulated MnO nanoparticles showed excellent stability in

various aqueous solutions, including physiological saline solu-

tion and human blood serum. Furthermore, by virtue of their

magnetic properties, MnO@SiO2–PEG/NH2 nanoparticles

showed a considerable T1 MR contrast enhancement, and since

a fluorescent dye was also incorporated into the silica shell during

the synthesis, our silica coated MnO nanoparticles may serve as

multimodal imaging agents for both optical and MR imaging. It

is worth mentioning that the SiO2 coated MnO particles (which

have an improved chemical stability compared to MnO particles

functionalized with non-innocent catechol) have relaxivities that

are comparable to those for the uncoated system. We also

demonstrated that silica encapsulated MnO nanoparticles are

less prone to manganese leaching in aqueous solution, compared

to simple PEGylated nanoparticles. Finally, the vast and well-

established silane chemistry permits additional modification of

the MnO@SiO2–PEG/NH2 nanoparticles, which leads to

numerous other applications, that is also confirmed by their low

toxicity and inert behaviour on primary immune cells.

Acknowledgements

We are grateful to the Center for Complex Matter (COMATT)

for support and thank Elisabeth Sehn for her skillful technical

support. K.K. was a recipient of a fellowship of the Graduate

School of Materials Science in Mainz of the State of Rhineland-

Palatinate, and T. D. Schladt was a recipient of a Carl-Zeiss

Fellowship. The Electron Microscopy Center in Mainz (EZMZ)

is operated through the Center for ComplexMatter (COMATT).

Notes and references

1 J. Cheon and J.-H. Lee, Acc. Chem. Res., 2008, 41, 1630–1640.2 T. D. Schladt, K. Schneider, H. Schild and W. Tremel, Dalton Trans.,2011, 40, 6315–6343.

3 M. I. Shukoor, F. Natalio, M. N. Tahir, V. Ksenofontov,H. A. Therese, P. Theato, H. C. Schr€oder, W. E. G. M€uller andW. Tremel, Chem. Commun., 2007, 4677–4679.

4 C. Sun, J. S. H. Lee andM. Zhang, Adv. Drug Delivery Rev., 2008, 60,1252–1265.

5 H. C. Schr€oder, F. Natalio, M. Wiens, M. N. Tahir, M. I. Shukoor,W. Tremel, S. I. Belikov, A. Krasko and W. E. G. M€uller, Mol.Immunol., 2008, 45, 945–953.

6 J. Gao, H. Gu and B. Xu, Acc. Chem. Res., 2009, 42, 1097–1107.7 Y.-W. Jun, J.-H. Lee and J. Cheon, Angew. Chem., 2008, 120, 5200–5213; Angew. Chem., Int. Ed. 2008, 47, 5122–5135.

8 J. H. Lee, Y. W. Jun, S. I. Yeon, J. S. Shin and J. Cheon, Angew.Chem., Int. Ed., 2006, 45, 8160–8162.

9 J.-L. Bridot, A.-C. Faure, S. Laurent, C. Rivi�ere, C. Billotey, B. Hiba,M. Janier, V. Josserand, J.-L. Coll, L. V. Elst, R. Muller, S. Roux,P. Perriat and O. Tillement, J. Am. Chem. Soc., 2007, 129, 5076–5084.

10 J. Y. Park, M. J. Baek, E. S. Choi, S. Woo, J. H. Kim, T. J. Kim,J. C. Jung, K. S. Chae, Y. Chang and G. H. Lee, ACS Nano, 2009,3, 3663–3669.

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

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

13 H. B. Na, I. C. Song and T. Hyeon,Adv. Mater., 2009, 21, 2133–2148.14 H. B. Na, J. H. Lee, K. An, Y. I. Park, M. Park, S. Lee, D. H. Nam,

S. T. Kim, S. H. Kim, S. W. Kim, K. H. Lim, K. S. Kim, S. O. Kim

J. Mater. Chem., 2012, 22, 9253–9262 | 9261

Dow

nloa

ded

by U

nive

rsity

of

Mas

sach

uset

ts -

Am

hers

t on

27 S

epte

mbe

r 20

12Pu

blis

hed

on 3

0 M

arch

201

2 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/C

2JM

1532

0C

View Online

and T. Hyeon, Angew. Chem., 2007, 119, 5341–5345; Angew. Chem.,Int. Ed., 2007, 46, 5397–5401.

15 H. B. Na and T. Hyeon, J. Mater. Chem., 2009, 19, 6267–6273.16 T. D. Schladt, K. Schneider, M. I. Shukoor, F. Natalio, H. Bauer,

M. N. Tahir, S. Weber, L. M. Schreiber, H. C. Schr€oder,W. E. G. M€uller and W. Tremel, J. Mater. Chem., 2010, 20, 8297–8904.

17 J. Huang, J. Xie, K. Chen, L. Bu, S. Lee, Z. Cheng, X. Li and X. Chen,Chem. Commun., 2010, 46, 6684–6686.

18 M. J. Baek, J. Y. Park, W. Xu, K. Kattel, H. G. Kim, E. J. Lee,A. K. Patel, J. J. Lee, Y. Chang, T. J. Kim, J. E. Bae, K. S. Chaeand G. H. Lee, ACS Appl. Mater. Interfaces, 2010, 2, 2949–2955.

19 M. E. Bennewitz, T. C. Lobo, M. K. Nkansah, G. Ulas,G. W. Brudwig and E. M. Shapiro, ACS Nano, 2011, 5, 3438–3446.

20 T. D. Schladt, T. Graf and W. Tremel, Chem. Mater., 2009, 21, 3183–3190.

21 J. Park, J. Joo, S. G. Kwon, Y. Jang and T. Hyeon, Angew. Chem.,2007, 119, 4714–4745; Angew. Chem., Int. Ed. 2007, 46, 4630–4660.

22 A.-H. Lu, E. Salabas and F. Sch€uth, Angew. Chem., 2007, 119, 1242–1266; Angew. Chem., Int. Ed. 2007, 46, 4630–4660.

23 N. A. Frey, S. Peng, K. Cheng and S. Sun, Chem. Soc. Rev., 2009, 38,2532–2542.

24 H. Goesmann and C. Feldmann, Angew. Chem., 2010, 122, 1402–1437; Angew. Chem., Int. Ed. 2010, 49, 1362–1395.

25 C. C. Berry and A. S. G. Curtis, J. Phys. D: Appl. Phys., 2003, 36,R198–R206.

26 S. M. Moghimi, A. C. Hunter and J. C. Murray, Pharmacol. Rev.,2001, 53, 283–318.

27 C. Xu, K. Xu, H. Gu, R. Zheng, H. Liu, X. Zhang, Z. Guo and B. Xu,J. Am. Chem. Soc., 2004, 126, 9938–9939.

28 V. P. Torchilin, Adv. Drug Delivery Rev., 2006, 58, 1532–1555.29 M. A. Dobrovolskaia, P. Aggarwal, J. B. Hall and S. E. McNeil,Mol.

Pharmaceutics, 2008, 5, 487–495.30 E. Amstad, S. Zurcher, A. Mashaghi, J. Y. Wong, M. Textor and

E. Reimhult, Small, 2009, 5, 1334–1342.31 J. Xie, C. Xu, N. Kohler, Y. Hou and S. Sun, Adv. Mater., 2007, 19,

3163–3166.32 M. Shukoor, F. Natalio, N. Metz, N. Glube, M. Tahir, H. Therese,

V. Ksenofontov, P. Theato, P. Langguth, J. P. Boissel,H. C. Schr€oder, W. E. G. M€uller and W. Tremel, Angew. Chem.,2008, 120, 4826–4830; Angew. Chem., Int. Ed. 2008, 47, 4748–4752.

33 T. D. Schladt, M. I. Shukoor, K. Schneider, M. N. Tahir, F. Natalio,I. Ament, J. Becker, F. D. Jochum, S. Weber, O. K€ohler, P. Theato,L. M. Schreiber, C. S€onnichsen, H. C. Schr€oder, W. E. M€uller andW. Tremel, Angew. Chem., 2010, 122, 4068–4072; Angew. Chem.,Int. Ed. 2010, 49, 3976–3980.

34 W. W. Yu, E. Chang, C. M. Sayes, R. Drezek and V. L. Colvin,Nanotechnology, 2006, 17, 4483–4487.

35 B.-S. Kim, J.-M. Qiu, J.-P. Wang and T. Taton, Nano Lett., 2005, 5,1987–1991.

36 S. K. Basiruddin, A. Saha, N. Pradhan and N. R. Jana, J. Phys.Chem. C, 2010, 114, 11009–11017.

37 S. Santra, H. Yang, P. H. Holloway, J. T. Stanley and R. A. Mericle,J. Am. Chem. Soc., 2005, 127, 1656–1657.

38 V. Salgueiri~no-Maceira,M.A.Correa-Duarte,M. Spasova, L.M.Liz-Marz�an and M. Farle, Adv. Funct. Mater., 2006, 16, 509–514.

39 H. Yang, S. Santra, G. A. Walter and P. H. Holloway, Adv. Mater.,2006, 18, 2890–2894.

40 H. Ow, D. R. Larson, M. Srivastava, B. A. Baird, W. W. Webb andU. Wiesner, Nano Lett., 2005, 5, 113–117.

41 A.Burns,H.OwandU.Wiesner,Chem.Soc.Rev., 2006,35, 1028–1042.42 E. Rampazzo, S. Bonacchi, M.Montalti, L. Prodi and N. Zaccheroni,

J. Am. Chem. Soc., 2007, 129, 14251–14256.

9262 | J. Mater. Chem., 2012, 22, 9253–9262

43 O.-H. Kim, S.-W. Ha, J. I. Kim and J.-K. Lee, ACS Nano, 2010, 4,3397–3405.

44 (a) L. M. Liz-Marz�an, M. Giersig and P. Mulvaney, Langmuir, 1996,12, 4329–4335; (b) S. Santra, R. Tapec, N. Theodoropoulou,J. Dobson, A. Hebard and W. Tan, Langmuir, 2001, 17, 2900–2906.

45 M. Bruchez, Jr, M. Moronne, P. Gin, S. Weiss and A. Alivisatos,Science, 1998, 281, 2013–2016.

46 Y. Kobayashi, M. A. Correa-Duarte and L. M. Liz-Marz�an,Langmuir, 2001, 17, 6375–6379.

47 A. Schroedter and H. Weller, Angew. Chem., 2002, 114, 3346–3350;Angew. Chem., Int. Ed. 2002, 41, 3218–3221.

48 Y. Piao, A. Burns, J. Kim, U. Wiesner and T. Hyeon, Adv. Funct.Mater., 2008, 18, 3745–3758.

49 A. Guerrero-Mart�ınez, J. P�erez-Juste and L. M. Liz-Marz�an, Adv.Mater., 2010, 22, 1182–1195.

50 H. Yang, Y. Zhuang, H. Hu, X. Du, C. Zhang, X. Shi, H. Wu andS. Yang, Adv. Funct. Mater., 2010, 20, 1733–1741.

51 Y.-K. Peng, C.-W. Lai, C.-L. Liu, H.-C. Chen, Y.-H. Hsiao,W.-L. Liu, K.-C. Tang, Y. Chi, J.-K. Hsiao, K.-E. Lim, H.-E. Liao,J.-J. Shyue and P.-T. Chou, ACS Nano, 2011, 5, 4177–4187.

52 T. Kim, E. Momin, J. Choi, K. Yuan, H. Zaidi, J. Kim, M. Park,N. Lee, M. T. McMahon, A. Quinones-Hinojosa, J. W. M. Bulte,T. Hyeon and A. A. Gilad, J. Am. Chem. Soc., 2011, 133, 2955–2961.

53 Y. Chan, J. Zimmer, M. Stroh, J. Steckel, R. Jain and M. Bawendi,Adv. Mater., 2004, 16, 2092–2097.

54 Y. Yang and M. Y. Gao, Adv. Mater., 2005, 17, 2354–2357.55 D. C. Lee, F. V. Mikulec, J. M. Pelaez, B. Koo and B. A. Korgel, J.

Phys. Chem. B, 2006, 110, 11160–11166.56 H. Yang, Y. Zhuang, H. Hu, X. Du, C. Zhang, X. Shi, H. Wu and

S. Yang, Adv. Funct. Mater., 2010, 20, 1733–1741.57 W. St€ober, A. Fink and E. Bohn, J. Colloid Interface Sci., 1968, 26,

62–69.58 C. Tessandier, N. Diop, M. Martini, S. Roux, O. Tillment and

T. Hamaide, Langmuir, 2012, 28, 209–218.59 (a) C. Louis, R. Bazzi, C. A. Marquette, J.-L. Bridot, S. Roux,

G. Ledoux, B. Mercier, L. Blum, P. Perriat and O. Tillement,Chem. Mater., 2005, 17, 1673–1682; (b) C.-W. Lu, Y. Hung,J.-K. Hsiao, M. Yao, T.-H. Chung, Y.-S. Lin, S.-H. Wu, S.-C. Hsu,H.-M. Liu, C.-Y. Mou, C.-S. Yang, D.-M. Huang and Y.-C. Chen,Nano Lett., 2007, 7, 149–154.

60 M. I. Shukoor, F. Natalio, P. Gupta, M. Wiens, M. Tarantola,M. Barz, S. Weber, M. Terekhov, H. C. Schr€oder, W. E. G. M€uller,A. Janshoff, P. Theato, R. Zentel, L. M. Schreiber and W. Tremel,Adv. Funct. Mater., 2009, 9, 3717–3725.

61 N. R. Jana, Y. Chen and X. Peng,Chem.Mater., 2004, 16, 3931–3935.62 T. Warger, P. Osterloh, G. Rechtsteiner, M. Fassbender, V. Heib,

B. Schmid, E. Schmitt, H. Schild and M. P. Radsak, Blood, 2006,108, 544–550.

63 M. P. Radsak, H. R. Salih, H.-G. Rammensee and H. Schild, J.Immunol., 2004, 172, 4956–4963.

64 H. Tan, J. M. Xue, B. Shuter, X. Li and J. Wang, Adv. Funct. Mater.,2010, 20, 722–731.

65 V. Mersseman, K. Besold, M. J. Reddehase, U. Wolfrum, D. Strand,B. Plachter and S. Reyda, J. Gen. Virol., 2008, 89, 369–379.

66 M. Ghosh, K. Biswas, A. Sundaresan and C. N. R. Rao, J. Mater.Chem., 2006, 16, 106–111.

67 W. S. Seo, H. H. Jo, K. Lee, B. Kim, S. J. Oh and J. T. Park, Angew.Chem., 2004, 116, 1135–1137; Angew. Chem., Int. Ed. 2004, 43, 1115–1118.

68 S. Santra, H. Yang, D. Dutta, J. T. Stanley, P. H. Holloway, W. Tan,B. M. Moudgil and R. A. Mericle, Chem. Commun., 2004, 2810–2811.

69 T. Bopp, M. P. Radsak, E. Schmitt and H. Schild, Cancer Immunol.Immunother., 2010, 59, 1443–1448.

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