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Fe3O4–silica core–shell nanoporous particles for high-capacity pH-triggereddrug delivery

X. F. Zhang,ab S. Mansouri,a L. Clime,a H. Q. Ly,d L. ’H. Yahiac and T. Veres*ab

Received 21st March 2012, Accepted 6th May 2012

DOI: 10.1039/c2jm31749d

We demonstrate a one-step procedure for the synthesis of Fe3O4–silica core–shell nanoparticles with

hierarchically ultra-large pores independent of any post-treatment such as annealing and template-

molecule removal. The nanoporous silica shells with available amine groups were functionalized by

clickable linkers to produce pH-sensitive amides for regulating the release of an anti-cancer drug,

doxorubicin (DOX). The loading amount of DOX reached up to 13.2 mg per 100 mg nanoparticles,

74.2% of which can be effectively released after 63 h at body temperature and pH 5 with decreased side

effects. Such excellent features of these nanoparticles appear to arise from the integrated hierarchically

ultra-large open-porosities and a homogeneous dispersibility in aqueous solution that has a great

potential for their use as drug delivery systems.

1 Introduction

In recent years, magnetic nanoparticles have been mainly studied

for their potential applications in a wide range of biomedical

fields, such as magnetic resonance imaging (MRI), targeted drug

delivery and magnetic separation. Currently, critical issues to be

resolved are their stability and biocompatibility in the circulatory

system, and surface functionalizations that conjugate the tar-

geting spacers or therapeutic agents.1,2 Core–shell structures have

been proposed in an effort to address the biochemical stability

and biocompatibility issues, as well as to provide a template

surface for the assembly of heterogeneous functions.3–5 Among

all the potential candidates, nanoporous shells provide the

distinct advantage of intrinsically higher surface areas, which are

especially important when employed as drug carriers.6�16

In the present work, we report on a one-pot procedure for the

synthesis of superparamagnetic core–shell Fe3O4–silica(porous)

nanoparticles containing both amine groups in the hierarchically

ultra-large porous silica shell. Compared with other synthesis

procedures for porous silica structure,6�16 the porous silica shell

in our case, with the pore size up to 10 nm, is in situ formed

without any post-treatment. The hierarchically ultra-large pores

ensure opened channels allowing a large-loading storage of guest

molecules, while also acting as interceptors for slow diffusion of

aBiomedical Engineering Department, McGill University, Quebec, CanadaH3A 2B4bIndustrial Materials Institute, National Research Council of Canada, 75Boul. de Mortagne, Boucherville, Qu�ebec, Canada J4B 6Y4. E-mail:Teodor.Veres@cnrc-nrc.gc.ca; Fax: +1 450 641-5105c �Ecole Polytechnique de Montr�eal, Case Postale 6079, succursale Centre-Ville, Montr�eal, Qu�ebec, Canada H3C 3A7dDepartment of Cardiovascular Medicine, Montreal Heart Institute,University of Montreal School of Medicine, Montreal, Quebec, Canada

This journal is ª The Royal Society of Chemistry 2012

these molecules due to the disordered topology of pore channels.

These advantages make these nanoparticles promising candi-

dates for biomolecule delivery. By functionalizing the nano-

particles with 1,2-cyclohexanedicarboxylic anhydride as a click

linker, we herein present the feasibility of coupling doxorubicin

(DOX) via amides to the porous silica shells with a superior

loading capacity up to 11.7 wt%. The coupled DOX molecules

are relatively stable at neutral pH 7.4, but can be rapidly

released in the range of pH 5.0–6.0 due to the hydrolysis of

amide bonds.

2 Experimental and theoretical sections

2.1 Materials

All reagents used in this study are commercially available. Oleic

acid (OA, 90%), 1-hexanol anhydrous (99%), octyl ether (98%),

ammonia solution (NH4OH, 28–30 wt% in water), Triton X-

100, hexane (95%), cyclohexane (99.5%), dimethyl sulfoxide

(DMSO, 99%), 1,2-cis-cyclohexanedicarboxylic anhydride

(98%), triethylamine (98%), tetraethoxysilane (TEOS, 99.999%),

sodium hydroxide (99%), tetrachloroaurate(III) hydrate

(99.99%), and DOX hydrochloride (98%) were purchased

from Sigma-Aldrich Inc. Iron pentacarbonyl (99.5%) was

purchased from Strem Chemicals, Inc. (Newburyport, MA) and

N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3), 3-

mercaptopropyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyl-

trimethoxysilane, N-ethylaminoisobutyltrimethoxysilane, N-

phenylaminopropyltrimethoxysilane, n-butylaminopropyl-

trimethoxysilane, tetraethoxysilane, and N-[(trimethoxysilyl)

propyl]poly(ethylenimine) were purchased from Gelest (Tully-

town, PA). Fluorescamine was purchased from MP Biomedi-

cals, LLC.

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2.2 Synthesis of core–shell Fe3O4–silica(nanoporous)

nanoparticles

The oleic acid-coated Fe3O4 (Fe3O4–OA) nanoparticles were

synthesized based on a well-known process.17 The core–shell

Fe3O4–silica nanoparticles were fabricated by hydrolyzing TEOS

in a water-in-oil microemulsion that contains the Fe3O4–OA

nanoparticles as seeds. Briefly, Fe3O4–OA nanoparticles were

first dispersed in cyclohexane, at a concentration of 1 mg ml�1,

and then 0.5 ml of the Fe3O4-containing cyclohexane dispersion

were rapidly injected into a mixture of 1.77 g of Triton X-100,

1.6 ml of anhydrous 1-hexanol and 7 ml of cyclohexane under

a strong vortex for about 1 h. Subsequently, 0.5 ml of ammonia

solution (28–30% ammonia solution : water ¼ 1 : 4) were added

in the above solution and shaken for another 1 h. Finally, 25 ml of

TEOS were added, and the mixture was allowed to react for 24 h.

The as-fabricated product was separated by centrifugation at

9000 rpm, washed with ethanol, and the silica shells are nonpo-

rous state (Fe3O4–silica nanoparticles). To synthesize the Fe3O4–

silica(porous) nanoparticles, 25 ml of AEAP3 were injected into

the above reaction mixture for another 24 h, and then the

product was separated by the same procedures. To uncover the

mechanism for the formation of a nanoporous structure we

studied seven types of different silanes with various molecular

structures, which were used in the second hydrolysis step of the

process.

2.3 DOX loading and pH-regulated release

2 mg Fe3O4–silica(porous) nanoparticles were dissolved in 20 ml

DMSO, followed by sonicating for 30 min. Triethylamine

(100 ml) was subsequently added and magnetically stirred for 2 h.

The grafted nanoparticles were separated by centrifugation at

9000 rpm, and mildly washed by DMSO three times. The grafted

nanoparticles and DOX hydrochloride salt (1 mg) were dissolved

in 20 ml DMSO solution, and magnetically stirred for 2 h. In

order to remove the free DOX molecules, the DOX-coupled

nanoparticles were separated by centrifugation and mildly

washed by pH 7.4 phosphoric acidic buffer solutions three times.

The release of DOX from coupled Fe3O4–silica(porous) nano-

particles was carried out at 37 �C and at pH 7.4, 6.0 and 5.0

phosphoric acidic buffer solutions, respectively. The separated

supernatant solution was monitored by UV-Vis spectra.

2.4 Cell culture

DOX release and the cytotoxicity of the Fe3O4–silica(porous)

core–shell nanoparticles functionalised with DOX were evalu-

ated using adenocarcinomic human alveolar basal epithelial cells

(A549, American Type Culture Collection (ATCC), USA). The

medium used was Ham’s F-12 (ATCC, USA) supplemented with

penicillin (100 IU ml�1), streptomycin (100 mg ml�1) and 10%

fetal bovine serum (FBS). The cells were cultured at a density of

1� 105 cells per 1 ml of medium in 24-well culture plates at 37 �Cin a 5% CO2 atmosphere. After 20 h of culture, the medium in

the wells was replaced with a fresh medium containing Fe3O4–

silica(porous) core–shell nanoparticles (1, 5, 10 and 50 mg ml�1),

Fe3O4–silica(porous) core–shell nanoparticles functionalised

with DOX (1, 5, 10 and 50 mg ml�1) and DOX (0.1, 0.5 1, and 5 mg

ml�1), and was further cultured for 48 h. In control cultures, the

J. Mater. Chem.

cells were placed in a medium without nanoparticles at the same

cell density. The image-capture system consisted of a Nikon

Eclipse microscope equipped with a filter set that has a scan

range from 380–750 nm. Using a �20 objective, bright-field and

fluorescence images were captured with a computer-controlled

charged-coupled device (CCD) camera using the Simple PCI

software imaging.

2.5 In vitro cell viability

The cell viability test was carried out via the reduction of the

MTT reagent (Invitrogen). After 48 h of culture with the Fe3O4–

silica(porous) core–shell nanoparticles (1, 5 10, and 50 mg ml�1),

Fe3O4–silica(porous) core–shell nanoparticles functionalized

with DOX (1, 5, 10, and 50 mg ml�1) and free DOX (0.1, 0.5, 1,

and 5 mg ml�1), 100 ml of MTT dye solution (5 mg ml�1 in

phosphate buffer pH-7.4) was added to each well and incubated

for 4 h at 37 �C and 5% CO2. The medium was removed and

formazan crystals were solubilized with 150 ml of dime-

thylsulphoxide (DMSO). Absorbance of each well was read

using a spectrophotometer (Biotek, USA) at 540 nm and the

relative cell viability (%) related to control wells containing

cell culture medium without nanoparticles was calculated by

[A]test/[A]control � 100. Three replicates were measured, and the

results presented as mean � standard deviation.

2.6 Characterization methods

The size and morphology of nanoparticles were analyzed using

a Hitachi S-4700 transmission electron microscope (TEM)

operated at a voltage of 30 kV. The microstructure and

composition of the samples were characterized by high resolution

TEM (HRTEM), selected area electron diffraction (SAED), and

energy dispersive X-ray spectroscopy (EDS) on a JEOL 2010F

(200 kV) transmission electron microscope. TEM samples were

prepared by dropping 25 ml of particle dispersion in hexane on

amorphous carbon coated copper grids, and drying under

vacuum overnight. FTIR spectra were collected with a Nicolet

Fourier spectrophotometer at wavenumbers between 600 and

4000 cm�1. UV-Vis spectra were collected on a Perkin Elmer

Lambda 950 spectrometer. Magnetic measurements of major

hysteresis loops (MHLs) at different temperatures as well as

zero-field cooled (ZFC) magnetization processes were performed

with a Quantum Design PPMS model 6000 magnetometer.

3 Results and discussion

3.1 Morphology and structures

Fig. 1(a) and (b) show typical transmission electron microscopy

(TEM) images of as-synthesized Fe3O4–OA (OA ¼ oleic acid)

nanoparticles. The particles are seen to have a narrow size

distribution and form a self-assembled super-lattice. The

measurement of about 200 particles has shown that the particles

are essentially spherical in shape, with a mean diameter of

15.1 nm. The corresponding fast Fourier transform (FFT)

pattern [inset of Fig. 1(b)] in the region marked with a red square

has a symmetrical lattice, indicating the single crystalline nature

of the nanoparticles. Fig. 1(c) and (d) show the Fe3O4–silica

nanoparticles with dense shells and the statistic particle

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 (a) A self-assembly TEM image and (inset) the size distribution from more than 200 particles of Fe3O4–OA nanoparticles; (b) high-resolution

TEM image and (inset) the fast Fourier transform pattern corresponding to the red square; (c) and (d) TEM images of core–shell Fe3O4–silica

nanoparticles; and (e) and (f) TEM images of the Fe3O4–silica(porous) nanoparticles.

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diameters of 56.2 � 0.09 nm. Fig. 1(e) and (f) show TEM images

of Fe3O4–silica(porous) nanoparticles. The as-synthesized

nanoparticles are all spherical in shape with an average total

diameter of 65.5 � 0.06 nm. Compared with the structural

features of Fe3O4–silica nanoparticles, it is worth noting that all

the particles present nanoporous structures.

Fig. 2 (a) EDS element mapping of Fe3O4–silica(porous) nanoparticles;

(b) line analysis along the axis of a single Fe3O4–silica(porous) nano-

particle, and (c) high angle annular dark field (HAADF) image of

a Fe3O4–silica(porous) nanoparticle, indicating the hierarchically ultra-

large pores with bicontinuous channels.

This journal is ª The Royal Society of Chemistry 2012

Fig. 2(a) and (b) show the energy dispersive X-ray spectros-

copy (EDS) elemental mapping and the line-scan analysis of

the elemental distribution of iron, oxygen, silicon and nitrogen

along the axis of one nanoparticle. A core–shell feature can be

clearly observed, indicating that the core is rich in iron and

oxygen, while the shell is mainly made of silicon, oxygen and

nitrogen. Fig. 2(c) shows the high angle annular dark field

(HAADF) image of Fe3O4–silica(porous) nanoparticles,

providing compelling evidence for the nanoporous structures,

revealing the hierarchically ultra-large porosities with bicontin-

uous channels extended to the surface. Such excellent features

can significantly improve the storage space of the guest mole-

cules, as well as the slow diffusion ascribed to the internal steric

hindrance of the hierarchical channels, offering a distinct

advantage for their use as drug delivery vehicles.

To reveal the formation mechanism of nanoporous silica shells,

we carriedout a comprehensive study by using another seven types

of silanes with different molecular structures, as shown in Fig. 3.

Although the mechanism of particle structures (dense, porous or

hollow) affected by the various silanes is still not quite clear, we

herein would like to make a speculation, according to the current

experimental observations. In our two-step hydrolysis process of

silica shell formation, the hydrophobic Fe3O4–OA nanoparticles

were firstly activated by Triton X-100 molecules via polyethylene

oxide groups to disperse in aqueous reaction cells, which provided

the condition for the condensation of TEOS molecules on the

surface of Fe3O4 nanoparticles. Under the effect of adsorbed

Triton X-100 molecules, the hydrolyzed TEOS silanes could form

a ‘soft’ shell consisting of incompletely condensed silica fragments

and Triton X-100 molecules. These fragments would further react

with subsequently added silane molecules (such as AEAP3).

Depending on the steric hindrance of functional groups with

various charges and backbones (such as NH2(CH2)2NH(CH2)3–

for AEAP3) of silane molecules, the silica shells could be finally

transformed into dense, porous, or even hollow structures when

removing the Triton X-100 molecules by ethanol washing.

J. Mater. Chem.

Fig. 3 TEM images of Fe3O4–silica core–shell nanoparticles with different microstructures depending on the usage of various silanes as indicated: (a)

3-mercaptopropyltrimethoxysilane; (b) 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane; (c) N-ethylaminoisobutyltrimethoxysilane; (d) N-phenyl-

aminopropyltrimethoxysilane; (e) n-butylaminopropyltrimethoxysilane; (f) tetraethoxysilane; (g)N-[(trimethoxysilyl)propyl]poly(ethylenimine); and (h)

AEAP3. The scales in all images are 200 nm.Dow

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3.2 Magnetic properties

Fig. 4(a)–(c) show the major hysteresis loops (MHLs) and corre-

sponding enlargements of Fe3O4–OA, Fe3O4–silica and Fe3O4–

silica(porous) nanoparticles at 10 and 300 K. The as-synthesized

Fe3O4–OA nanoparticles, in Fig. 4(a), exhibit typical super-

paramagnetic behavior, consisting of Langevin-type curves of

nearly zero coercive fields at room temperature. As expected, in

the low temperature regime, blocked (ferromagnetic) particles

become preponderant and the MHLs become slightly hysteretic,

with an increased saturation magnetization (Ms) of �73 emu g�1

and a coercive field (Hc) of �53 Oe. The chemical analysis results

from Guelph Chemical Laboratories Ltd. show that the as-

synthesized Fe3O4–OA nanoparticles contain about 62.5 wt%

iron, corresponding to 86.3 wt% Fe3O4 and 13.7 wt% OA

regardless of other impurities. This corresponds to a net value of

84.5 emu g�1 for normalized Fe3O4 nanoparticles, as previously

reported by Sun et al.18 However, the organic species can be

removed completelyon silica coating, as seen fromthe fastFourier

transmission infrared (FTIR) spectra. In Fig. 4(b) and (c), theMs

values of Fe3O4–silica and Fe3O4–silica(porous) nanoparticles

are �3.1 and �1.1 emu g�1, corresponding to the nonmagnetic

silica compositions of 96.3 wt% and 98.7 wt%, respectively.

J. Mater. Chem.

Fig. 4(d)–(f) show the temperature-dependent zero-field-cooling

(ZFC) andfield-cooling (FC)magnetization curves ofFe3O4–OA,

Fe3O4–silica and Fe3O4–silica(porous) nanoparticles, respec-

tively, measured at an applied magnetic field of 50 Oe. The ZFC

curve of Fe3O4–OA nanoparticles exhibits a broader maximum

of �200 K (Tmax). The Tmax values of Fe3O4–silica and Fe3O4–

silica(porous) nanoparticles become more obvious and shift to

lower temperatures at 109 and 101 K, respectively, as the thick-

nesses of shells increase, although the Fe3O4 cores were not

changed. For isolated, non-interacting nanoparticles, Tmax is

normally related to the blocking temperature (TB) at which the

particles undergo a phase transition from ferromagnetic to

superparamagnetic. As for the ZFC analysis, the experimental

curveswere compared toa theoreticalmodel basedon theblocking

behavior of assemblies of superparamagnetic nanoparticles.19

3.3 Surface chemistry

In order to confirm the functional groups on the surface of

nanoparticles, the FTIR spectra were collected on (a): Fe3O4–

OA, (b): Fe3O4–silica and (c): Fe3O4–silica(porous) nano-

particles, as shown in Fig. 5. The absence of –OH vibrations

This journal is ª The Royal Society of Chemistry 2012

Fig. 4 (a–c) Hysteresis loops at 10 and 300 K of (a) Fe3O4–OA, (b) Fe3O4–silica and Fe3O4–silica(porous) nanoparticles after zero field cooling. (d–f)

ZFC-FCmagnetization curves of Fe3O4–OA, Fe3O4–silica and Fe3O4–silica(porous) nanoparticles under an applied magnetic field of 50 Oe. Insets in (a)

and (b) are the enlargements of the graph near the origin. The inset in (c) is the photograph of Fe3O4–silica(porous) nanoparticles dispersed in DI water,

with and without magnetic separation.

Fig. 5 FTIR spectra of (a) Fe3O4–OA, (b) Fe3O4–silica and (c) Fe3O4–silica(porous) nanoparticles.

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at �3300 cm�1 from acid groups indicates that all the OA

molecules have reacted with the Fe3O4 surface and no phys-

isorbed oleic acid molecules remained. This results in a self-

assembled oleate monolayer around the surface of Fe3O4

nanoparticles. Thus, the Fe3O4–OA nanoparticles bear –CH3

groups at the free termini of OA chains, leading to a hydrophobic

behavior. As shown in Fig. 5(b), asymmetric and symmetric

stretching vibrations of ^Si–O–Si^ were observed at about

1080 and 798 cm�1, respectively, corresponding to characteristic

peaks of silica.20,21 The peaks at 3400, 1637 and 960 cm�1 are

assigned, respectively, to the stretching and deformation vibra-

tions of adsorbed water molecules and the stretching mode

Si–OH (hydroxyl groups).22–24 The FTIR spectrum of Fe3O4–

silica(porous) nanoparticles in Fig. 5(c) shows that the

characteristic peaks of silica at 1050 (asymmetric stretching^Si–

O–Si^) and 920 (symmetric stretching ^Si–O–Si^) shift to

lower wavenumbers compared to that of Fe3O4–silica nano-

particles, which is ascribed to the effect of amino-

ethylaminopropyl groups. The broad peak at �3350 cm�1 is due

to an overlap of hydrogen-bonded O–H and N–H stretching.

The peaks at �2900 cm�1 are due to stretching vibrations

This journal is ª The Royal Society of Chemistry 2012

of –CH2– bonds. The multiple peaks between 1300 and 1600

cm�1 are consequences of the vibrations of amine groups.25 The

FTIR analyses confirmed that the silica shells of Fe3O4–sili-

ca(porous) nanoparticles are in situ functionalized by hydroxyls,

primary and secondary amine groups.

3.4 pH-triggered DOX release

DOX is one of the most widely used chemotherapeutic drugs.

However, it is limited by dose-dependent toxic side effects.26

Thus, targeted drug delivery, providing therapeutically effective

drug release at the tumor site, exhibits immense potential to

resolve this issue and improve the treatment of cancers.27,28 The

coupling and pH-sensitive hydrolysis properties of DOX mole-

cules with primary and secondary amine groups, via 1,2-cyclo-

hexanedicarboxylic anhydride as a linker, have been reported

previously.29,30 The amides with neighboring carboxylic acid

groups are stable at neutral pH, while at a low pH they are

negatively charged to regenerate the amine groups and release

the free DOX molecules. In line with this concept, we developed

a magnetically guided pH-triggered drug delivery carrier based

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on the Fe3O4–silica(porous) nanoparticles. The Fe3O4–sili-

ca(porous) nanoparticles with primary and secondary amine

groups were preliminarily functionalized by grafting 1,2-cyclo-

hexanedicarboxylic anhydride molecules. The other side of 1,2-

cyclohexanedicarboxylic anhydride molecules was subsequently

coupled to the amine groups of DOX molecules, forming the

same amide bonds with neighboring carboxylic acid groups.

Under normally physiological conditions (pH z 7), the coupled

DOX molecules are quite stable, whereas mild acid hydrolysis

can release them when they are transported in the vicinity of

cancerous tissues (pH 5–6).30–32

The characteristic peaks of DOX molecules at 450–550 nm in

UV-vis spectra are used to confirm the coupling and release

processes. Concentration of the released DOX was examined by

comparing the normalized absorbance intensity of separated

supernatant solutions. Based on the intensity at 504 nm, the

amount of coupled DOX was estimated at 13.2 mg per 100 mg

nanoparticles, while the released amount of DOX molecules, at

pH 5.0 for 63 h, was estimated to be about 9.8 mg per 100 mg

nanoparticles. As a comparison, we also carried out the

measurement of the loading capacity of DOX molecules in the

Fe3O4–silica nanoparticles with dense silica shells, confirming

that the maximum loading capacity is only 1.2 mg per 100 mg

nanoparticles. It is clearly indicated that, in the case of Fe3O4–

silica(porous) nanoparticles, DOXmolecules are loaded not only

on the surface of silica shells, but also on the interior pore/

channel surfaces. Fig. 6 shows the release kinetic of DOX

molecules as a function of the pH and release time. It indicates

that at a pH of 7.4 only 3.8% of the initial DOX molecules’

payload was released after 10 h. This low loss of the initial charge

replies that the amide bonds were stable enough in order to retain

the DOX molecules. As the pH decreased, a sharp increase was

measured in the concentration of the DOX molecules released in

the supernatant solution. The concentrations released after 10 h

at pH 6 and 5 were estimated to be 46% and 76% respectively.

These data revealed that the release process of DOX from the

Fe3O4–silica(porous) nanoparticles is pH-triggered and can be

well controlled.

Conversely, in Fig. 6, we see that the DOX release process

exhibits a bimodal release pattern. It seems that the early-time

window, from 0 to 3.25 h, has different dynamics than the late-

time interval, from 3.25 to 10 h, for both pH 6.0 and 5.0. Obvi-

ously, the origin of this bimodal behavior is related to two

Fig. 6 Release profiles of DOX molecules from Fe3O4–silica(porous)

nanoparticles in buffer solutions of pH 5.0, 6.0 and 7.4 at 37 �C.

J. Mater. Chem.

different release kinetics, that have different diffusion coeffi-

cients. A similar bimodal release behavior has been observed in

other pH-regulated nanocarriers such as porous silicon nano-

particles with a nanovalve system with a cyclodextrin cap33 and

mesoporous silica nanoparticles with ZnO nanolids.34 In these

systems, the release is mainly controlled by both the pH-depen-

dent hydrolysis kinetics and the diffusion (or dissolution) of

cyclodextrin nanovalves (or ZnO nanolids). In comparison, the

bimodal release behavior in the Fe3O4–silica(porous) nano-

particles indicates that the diffusion of released DOX molecules

could also be affected by the hierarchical architecture of nano-

channels. The hierarchical nanochannels act as interceptors to

slow the diffusion which is similar to the role of nanovalves. Such

a feature is important as the hierarchical porous carriers

proposed herein can release their cargo within a specific timescale

of interest.

3.5 Delivery of DOX-nanoparticles into cells

The therapeutic efficacies of Fe3O4–silica(porous) nanoparticles

functionalized with DOX were tested using A549 cells, adeno-

carcinomic human alveolar basal epithelial cells. After incuba-

tion for 48 h, an MTT assay was performed to evaluate the

viability of A549 cells, as shown in Fig. 7. These results indicate

that the Fe3O4–silica(porous) nanoparticles are relatively non-

toxic at lower concentrations (1, 5 and 10 mg ml�1) with around

70% cell viability, whereas both free DOX and Fe3O4–sili-

ca(porous)–DOX nanoparticles exhibited a significant loss of cell

viability, most notably at 50 mg ml�1. However, at the same DOX

concentration of 0.5, 1 and 5 mg ml�1, Fe3O4–silica(porous)–

DOX nanoparticles show higher viability compared with free

DOX. The cytotoxicity effect was further confirmed by micro-

scopic visualization of the cell morphology change after treat-

ment with the nanoparticles and/or DOX. Fig. 8 shows the

morphologies of A549 cells treated with Fe3O4–silica(porous)

nanoparticles, Fe3O4–silica(porous)–DOX nanoparticles and

free DOX, respectively. Cells incubated with DOX became

rounded and non-adherent, indicative of the fact that they have

undergone apoptosis. In contrast, no rounded and detached cells

can be visualized in both control cells and cells treated with

Fe3O4–silica(porous) and Fe3O4–silica(porous)–DOX nano-

particles. The loaded DOXmolecules are visualized hidden in the

Fig. 7 Cell viability percentage after 48 h of incubation with Fe3O4–

silica(porous), Fe3O4–silica(porous)–DOX nanoparticles, and free DOX

with various concentrations.

This journal is ª The Royal Society of Chemistry 2012

Fig. 8 Bright-field microscopic images of the cells incubated after 48 h with Fe3O4–silica(porous), Fe3O4–silica(porous)–DOX nanoparticles, and free

DOX with various concentrations.

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hierarchically ultra-large channels of the Fe3O4–silica(porous)

nanoparticles, which reduces their effective exposure to cells, and

therefore results in a lower toxic effect.

Fluorescence images were used to determine intracellular

localization and accumulation of DOX in A549 cells. DOX is

localized in their cytoplasm and subcellular compartment as

indicated by the clearly visible red fluorescence of DOX in Fig. 9.

However, rounded and non-adherent cells could be found in free

Fig. 9 (a and c) Bright-field and (b and d) fluorescence microscopic

images of DOX release detection of (a and b) 10 mg ml�1 Fe3O4–sili-

ca(porous)–DOX nanoparticles and (c and d) 1 mg mg�1 free DOX in cells

with incubation of 48 h.

This journal is ª The Royal Society of Chemistry 2012

DOX treated cells. This implies that the Fe3O4–silica(porous)–

DOX nanoparticles could be directly uptaken by cells with

reduced toxicity. Moreover, the uniform fluorescence intensity

and distribution in each cell indicates that the Fe3O4–silica(po-

rous)–DOX nanoparticles have a good dispersibility in the

medium and no aggregation occurs when interacted with cells,

which is attributed to a combined consequence of the nearly zero

magnetic interaction and the surface charged hydroxyls.

4 Conclusions

We report a one-pot synthesis for producing water soluble,

amino-functionalized Fe3O4–silica(porous) core–shell nano-

particles with a 15.1 nm Fe3O4 core and a 20 nm nanoporous

silica shell. The porous silica shell was characterized by high

angle annular dark field imaging technology, revealing the hier-

archically ultra-large pores and their channels extended to the

surface. We further developed and demonstrated a chemical

protocol to covalently couple and release DOX via dual regula-

tions of the pH-dependent hydrolysis kinetics of the amides and

the hierarchical nanochannels that act as interceptors for slow

diffusion. The largest amount of released DOX at pH 5 after 63 h

was about 9.8 mg for 100 mg Fe3O4–silica(porous) nanoparticles,

and 76% of them can be effectively released after 10 h. At pH 7.4,

only 3.8% and 9% were released after 10 and 63 h, respectively.

The nanoparticles synthesized herein show a saturated magne-

tization of 1.1 emu g�1. Based on a theoretical model, the

temperature-dependent magnetization processes point toward

a nearly zero magnetic interaction between superparamagnetic

Fe3O4 cores due to the steric hindrance of the shells, contributing

to good dispersibility. Such significant features meet the desirable

requirements for a drug delivery system while providing the

possibility of effectively tracking the drug carriers using MRI

technology.

J. Mater. Chem.

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Acknowledgements

The work was jointly supported by the NSERC-CRD grant, the

Canadian Institutes of Health Research and the National

Research Council of Canada, Industrial Materials Institute

(IMI-NRC). The authors would like to thank Francois Nor-

mandin from IMI-NRC for the help with magnetic measure-

ments and Dr Gianluigi Botton from McMaster University

Center for Electron Microscopy for his help with the TEM

characterization. We are grateful to Nitric Medical Devices Inc.

for the financial support and to Dr Blaise Gilbert and Dr Omar

Quraishi for the insightful advice for the use of the magnetic

carriers for biomedical applications. We would like to thank

Dr Edward Sacher for technical revision of the manuscript.

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