Materials Research Bulletin 59 (2014) 65–68

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Materials Research Bulletin

journal homepage: www.elsevier.com/locate/matresbu

Mesoporous Fe3O4/hydroxyapatite composite for targeted drug

delivery
Lina Gu, Xiaomei He, Zhenyu Wu *
School of Chemistry and Chemical Engineering, Anhui University, Hefei, Anhui 230039, China

A R T I C L E I N F O A B S T R A C T

Article history: In this contribution, we introduced a simple, efficient, and green method of preparing a mesoporous
Received 28 February 2014 Fe3O4/hydroxyapatite (HA) composite. The as-prepared material had a large surface area, high pore
Received in revised form 4 June 2014 volume, and good magnetic separability, which made it suitable for targeted drug delivery systems. The
Accepted 14 June 2014
chemotherapeutic agent doxorubicin (DOX) was used to investigate the drug release behavior of Fe3O4/
Available online 19 June 2014
HA composite. The drug release profiles displayed a little burst effect and pH-dependent behavior. The
release rate of DOX at pH 5.8 was larger than that at pH 7.4, which could be attributed to DOX protonation
Keywords:
in acid medium. In addition, the released DOX concentrations remained at 0.83 and 1.39 mg/ml at pH 7.4
A. Inorganic compounds
A. Magnetic materials
and 5.8, respectively, which indicated slow, steady, and safe release rates. Therefore, the as-prepared
B. Chemical synthesis Fe3O4/hydroxyapatite composite could be an efficient platform for targeted anticancer drug delivery.
D. Surface properties ã 2014 Published by Elsevier Ltd.

1. Introduction
Conventional dosage forms (e.g., oral medicine and injection)
have toxic and side effects on healthy tissues and exhibit peak-to-
through plasma concentration. Time-release dosage is an effective
solution, it is a formulation of drug delivery system that controls
the rate and period of drug delivery [1–6]. Among the variety of
drug delivery systems that have been developed, magnetic drug
delivery is an efficient method to selectively deliver a drug to a
targeted pathological site in the body [7]. By application of an
external magnetic field, the magnetic material, together with the
drug molecules, can be targeted to the nidus and only react on this
location. Meanwhile, controlled drug release could achieve a stable
plasma concentration and reduce drug times.
Given the advantages of easy synthesis, small size, low toxicity,
and unique superparamagnetism, superparamagnetic magnetite
nanoparticles (NPs) are widely applied to targeted delivery
systems. These NPs can be incorporated within the drug carrier
systems to facilitate the manipulation and delivery of the drug-
loaded nano-carriers toward a desired area by externally localized
magnetic steering. Therefore, drug and gene delivery by super-
paramagnetic magnetite NPs have great clinical potential [8–10].
As a drug delivery system, the drug carrier should release the
drug at a suitable and stable rate. However, burst effect is often
observed. Very small amounts of drug can reach the required site
* Corresponding author. Tel.: +86 551 63861326; fax: +86 551 63861279.
E-mail address: zhenyuwuhn@sina.com (Z. Wu).
because the majority of the loaded drug is rapidly released at the
initial stage. To reduce the burst effect, Mahmoudi et al. [11]
prepared iron oxide NPs with a crosslinked poly(ethylene glycol)-
co-fumarate coating. The result showed that the burst release is
reduced by 21% compared with the non cross-linked particles. Ke
et al. [12] synthesized Fe3O4/Cu3(BTC)2 nanocomposite by
incorporating Fe3O4 nanorods with Cu3(BTC)2 nanocrystals. An
early rapid release of 20% occurred within 4 h, followed by a slow
and steady release after more than 11 days. In this study,
mesoporous Fe3O4/hydroxyapatite (HA) composite was synthe-
sized by a combination of precipitation and hydrothermal process.
This process is a simple, efficient, and green approach to prepare
magnetic drug delivery materials. The as-prepared composite
possessed large specific surface area (SSA), high pore volume, and
magnetic property. These characteristics make it a good candidate
for targeted drug release materials. Drug loading and pH-
controlled release of Fe3O4/HA composite were investigated using
doxorubicin (DOX) as a model drug. The results demonstrated that
DOX-loaded Fe3O4/HA composite can effectively avoid burst effect
and give a steady and long-term release.
2. Experimental
2.1. Materials
Chemicals were used as received, including Ca(NO3)2
4H2O,
(NH4)2HPO4, (NH4)Fe(SO4)2
12H2O, FeSO4
7H2O and NH4OH (25%).
These chemicals were obtained from Tianjin Guangfu Fine
Chemical Research Institute. Absolute ethanol was purchased

http://dx.doi.org/10.1016/j.materresbull.2014.06.018
0025-5408/ ã 2014 Published by Elsevier Ltd.
66 L. Gu et al. / Materials Research Bulletin 59 (2014) 65–68
from Shanghai Zhenxing First Chemical Industry Factory. DOX was
obtained from Yongnuo pharmaceutical factory. All of the reagents
were of analytical purity.
2.2. Preparation of Fe3O4/HA composite
Fe3O4 magnetic NPs were synthesized by gas–liquid chemical
precipitation combined with hydrothermal method. (NH4)Fe
(SO4)2
12H2O (0.964 g, 2 mmol) and FeSO4
7H2O (0.278 g, 1 mmol)
were separately dissolved in distilled water/glycerol (50 ml, 9:1, v/
v) solution. As shown in Fig. 1a, a beaker containing a
stoichiometric ratio of (NH4)Fe(SO4)2 and FeSO4 solutions was
placed into a sealed bigger beaker of ammonia solution. The
mixture was stirred for 2 h at room temperature. The resulting
mixture was then transferred to a Teflon-lined stainless-steel
autoclave and heated at 120  C for 12 h. The precipitate was washed
with distilled water and anhydrous ethanol and dried at 60  C in
vacuum. Fe3O4 (0.1 g) was dispersed in alcohol solution of Ca
(NO3)2
4H2O (12.5 mmol, 50 ml) to obtain suspension. The
suspension beaker was placed into a sealed bigger beaker filled
with ammonia solution. Under continuous stirring, the aqueous
solution of (NH4)2HPO4 (7.5 mmol, 50 ml) was added dropwise to
the suspension (Fig. 1b). Then, the mixture was stirred for 12 h and
stored for another 12 h. The product was separated by filtration and
washed with distilled water and anhydrous ethanol before
vacuum-dried. Without the addition of Fe3O4, pure HA could be
obtained.
2.3. Characterization
The phase analysis was carried out by Purkinje XD-3 powder X-
ray diffracmeter (XRD) with a Rigaku D/max-gA rotation anode. To
examine the size and morphology of the as-synthesized samples,
TEM images were taken with a JEOL JEM-2100 high resolution
electron microscopy using an accelerating voltage of 200 kV. SSA
and pore size distribution (PSD) were determined using MICRO-
MERITICS ASAP2020M+C surface area and porosity analyzer with a
degas temperature of 105  C and an outgas time of 12 h according
to the Brunauer–Emmett–Teller (BET) equation and the method of
Barrett–Joyner–Halenda (BJH), respectively. The magnetization
curves were measured at room temperature under a varying
magnetic field of 1,0000 to 10,000 Oe on a BHV-55 vibrating
sample magnetometer.
2.4. Drug loading and in vitro drug release
A total of 10 mg of Fe3O4/HA was added into the DOX aqueous
solution (5 ml, 700 ug/ml). The resulted suspension was shaken
(180 rpm) in a shaking bed (HQ45B, Wuhan Science and
Technology Instrument Factory) for 24 h. After equilibrium, the
Fig. 1. Schematic illustration of preparing (a) Fe3O4 and (b) Fe3O4/HA composite.
liquid and solid phases were separated by centrifugation.
Ultraviolet–visible (UV–vis) spectrophotometry (Purkinje TU-
1901 model UV–vis double beam spectrophotometer) was used
to measure DOX concentration at 480 nm. In vitro release of DOX
from Fe3O4/HA composite was carried out in PBS with pH 7.4 and
5.8. Briefly, 25 mg of DOX-loaded Fe3O4/HA composite was
immersed into the release medium (25 ml). The vials were shaken
at 180 rpm at 37  C in a shaking bed. At predetermined time
intervals, after the dispersion was magnetically separated, 6 ml of
the release medium was obtained for measurement and replaced
with an equal volume of fresh phosphate buffer solution.
3. Results and discussion
3.1. Characterizations of Fe3O4/HA composite
Fe3O4 and Fe3O4/HA composite were synthesized by a
combination of gas–liquid chemical preparation and hydrothermal
method. XRD was carried out to characterize the crystalline phases
of the products. Fig. 2a shows that the positions and relative
intensities of the diffraction peaks could be well indexed to a cubic
inverse structure (JCPDS card 19-0629), indicating the formation of
Fe3O4 crystal structure. All characteristic diffraction peaks of Fe3O4
also appeared in the XRD pattern of Fe3O4/HA (Fig. 2b, marked with
“+”), which confirmed the existence of Fe3O4 in Fe3O4/HA
composite. Other peaks in Fig. 2b corresponds to the hexagonal
HA (Fig. 2c, JCPDS card 09-0432). The TEM images of Fe3O4, HA, and
Fe3O4/HA composite are presented in Fig. 3a suggests that Fe3O4
were uniform NPs with a diameter of 10 nm–20 nm and possessed
disordered ultramicroporous. Fig. 3b shows the TEM image of HA
(without Fe3O4). The pure HA were well dispersed fusiform
particles with 20 nm in width and 100–150 nm in length.
Moreover, mesoporous structures with a pore size about 2 nm
appeared on the surface. After the addition of Fe3O4 (Fig. 3c), the
size of the HA particle increased a little, but its morphology
remained unchanged. Fe3O4 NPs were also well embedded in
mesoporous HA. The surface area, pore volume and pore size
distributions of Fe3O4, HA, and Fe3O4/HA composite were analyzed
by nitrogen adsorption–desorption techniques. Fig 4a shows that
the type IV isotherms with a distinct hysteresis loop indicated the
presence of mesopores in three compounds [13] BJH pore size
Fig. 2. XRD patterns of (a) Fe3O4, (b) Fe3O4/HA composite, (c) HA, (d) the standard
data for Fe3O4 (JCPDS No. 19-0629) and (e) the standard data for hydroxyapatite
(JCPDS No. 09-0432).
 L. Gu et al. / Materials Research Bulletin 59 (2014) 65–68 67

Fig. 3. TEM images of (a) Fe3O4, (b) HA and (c) Fe3O4/HA composite.

Fig. 4. (a) Nitrogen adsorption–desorption isotherms and (b) pore size distributions of Fe3O4, HA and Fe3O4/HA composite.

distributions (Fig. 4b) demonstrated that two kinds of mesopores
with sizes of 2–4 nm and 4–10 nm were observed. On the basis of
the pore sizes, we can deduce that pores around 2–4 nm were
formed in the particles whereas other larger pores were formed
among the particles. This result is in accordance with that obtained
from TEM technique. For the smallest size, Fe3O4 exhibited the
most inter-particle pores and the largest surface area (143.39 m2/
g). The calculated BET results showed that the addition of Fe3O4
increased the surface area of Fe3O4/HA (124.05 m2/g) compared
with that of pure HA (116.80 m2/g), which may be attributed to the
pores distributed on the Fe3O4 particles. These Fe3O4/HA particles
with large surface area and high pore volume (0.42 cm3/g) will be
an excellent candidate for drug delivery [14]. Superparamagnetic
property and high saturation magnetization (SM) values are the
two most important parameters for a magnetic targeted delivery
system [8]. Fig. 5a shows the magnetization curves of Fe3O4 and
Fe3O4/HA composite at room temperature. The SM of Fe3O4 and
Fe3O4/HA composite were 68.87 and 16.20 emu/g, respectively. The
HA coat reduced the weight ratio of Fe3O4 in NPs; thus, the SM of
the composite dropped significantly. Moreover, the hysteresis
loops (near-zero coercivity and remanence effect) showed super-
paramagnetic property of the Fe3O4/HA composite. The magnetic
separability of DOX-loaded Fe3O4/HA composite is shown in
Fig. 5b. Without an outer magnet, the Fe3O4/HA composite could
be well dispersed in DOX solution (left bottle). In the right bottle,
the composite could be rapidly gathered on the side of the bottle
when a magnet was placed near the bottle. This result directly
reveals that the DOX-loaded Fe3O4/HA composite had magnetic
properties and was suitable for application in targeted drug
delivery systems.
3.2. Release behavior of DOX-loaded Fe3O4/HA composite
According to the UV–vis spectra analysis of the residual
aqueous solution after the drug-Fe3O4/HA adsorption, the DOX
content was determined to be 0.102 g of DOX per gram of Fe3O4/HA
composite. Controlled drug release experiments were carried out
using PBS buffer solutions with pH 7.4 and 5.8 as the release media.

Fig. 5. (a) Magnetization curves of Fe3O4 and Fe3O4/HA composite at room temperature. (b) Separation of DOX-loaded Fe3O4/HA composite suspension before (left) and after
(right) being attracted by a magnet.
68 L. Gu et al. / Materials Research Bulletin 59 (2014) 65–68
Fig. 6. DOX release profiles of DOX-loaded Fe3O4/HA composite at pH 7.4 and 5.8 at
37  C.
The pH values were selected based on the physiological pH in the
blood stream (pH 7.4) [15] and endosomes (pH 5.5–6.4) [16]. Fig. 6
shows the DOX release process from the Fe3O4/HA composite in
PBS at 37  C. The drug release process could be divided into two
regions. At the early stage, a rapid release occurred within 20 h, and
about 13% and 20% of DOX were released at pH 7.4 and 5.8,
respectively, which were far less than the other targeted drug
delivery systems (e.g., 25% within 10 h at pH 7.4 for PMMNP [8] and
20% within 8 h at pH 7.4 for Fe3O4@SiO2 hollow mesoporous
spheres [17]). In region 2, a slow and steady release lasted for a very
long time: 28% and 43% were released in the next 100 h. The drug
release at pH 5.8 was greater than that at pH 7.4, which was similar
to the results observed in other magnetic DOX carriers [5,18]. This
behavior is attributed to DOX protonation in the acid medium
[19,20].
After the early rapid release stage, the DOX concentrations of
collected supernatant solution at predetermined time intervals
maintained at a certain value. It did not increase despite the
lengthened interval of obtaining the buffer solution. Once the
concentration of DOX decreased, the DOX-loaded Fe3O4/HA
composite could continue to release DOX until the DOX
concentration reached the limit. This fact reveals that the as-
prepared Fe3O4/HA composite could release DOX at a steady rate,
which is the only drug delivery system required. In addition, the
released concentrations of DOX were around 0.83 and 1.39 mg/ml
at pH 7.4 and 5.8, respectively. These concentrations were safe
compared with the upper DOX (10 mg/ml) for the dose–response
experiment [17]. Consequently, the DOX-loaded Fe3O4/HA com-
posite could not only avoid burst effect but also release drug at a
long-term, steady, and safe rate.
4. Conclusions
In summary, we successfully synthesized mesoporous Fe3O4/HA
composite and demonstrated its controlled drug release behavior.
The preparation process was simple, efficient and environmentally
friendly. The SM of the Fe3O4/HA composite was 16.20 emu/g, and the
drug-loaded composite showed magnetic separability well. The
burst effect of this material could be effectively avoided because less
than 20% was released within 20 h. This result is followed by a slow
and steady release after more than 100 h. Therefore, this material
could decrease drug times and reduce suffering of cancer patients,
which is what the targeted drug delivery system requires.
Acknowledgements
The authors are grateful for the financial support by the
National Natural Science Foundation of China(No. 21001001) and
the Doctoral Research Foundation of Anhui University
(No.02303319-33190097;33190184).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.materres-
bull.2014.06.018.
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