materials
Article
Preparation and Characterization of Thermoresponsive
Poly(N-isopropylacrylamide-co-acrylic acid)-Grafted
Hollow Fe3O4/SiO2 Microspheres with Surface Holes
for BSA Release
Jing Zhao 1 , Ming Zeng 1 , Kaiqiang Zheng 1 , Xinhua He 1 , Minqiang Xie 2 and Xiaoyi Fu 1, *
1
2
*
School of Materials Science and Engineering, South China University of Technology,
Guangzhou 510641, China; [email protected] (J.Z.); [email protected] (M.Z.);
[email protected] (K.Z.); [email protected] (X.H.)
Department of Otorhinolaryngology Head & Neck Surgery, Zhujiang Hospital of Southern Medical
University, Guangzhou 510282, China; [email protected]
Correspondence: [email protected]; Tel./Fax: +86-20-8711-0149
Academic Editor: Jordi Sort
Received: 6 March 2017; Accepted: 12 April 2017; Published: 14 April 2017
Abstract: Thermoresponsive P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres with surface holes serving
as carriers were prepared using p-Fe3 O4 /SiO2 microspheres with a thermoresponsive copolymer.
The p-Fe3 O4 /SiO2 microspheres was obtained using a modified Pickering method and chemical
etching. The surface pore size of p-Fe3 O4 /SiO2 microspheres was in the range of 18.3 nm~37.2 nm
and the cavity size was approximately 60 nm, which are suitable for loading and transporting
biological macromolecules. P(NIPAM-AA) was synthesized inside and outside of the p-Fe3 O4 /SiO2
microspheres via atom transfer radical polymerization of NIPAM, MBA and AA. The volume
phase transition temperature (VPTT) of the specifically designed P(NIPAM-AA)/Fe3 O4 /SiO2
microspheres was 42.5 ◦ C. The saturation magnetization of P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres
was 72.7 emu/g. The P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres were used as carriers to study the
loading and release behavior of BSA. This microsphere system shows potential for the loading of
proteins as a drug delivery platform.
Keywords: P(NIPAM-AA); Fe3 O4 /SiO2 hollow microspheres; holes; drug release
1. Introduction
Targeted drug delivery systems are a hot topic in the fields of biology and medicine [1,2].
In previous studies, the microspheres used as carriers usually have had a pore size of 2–3 nm, which
restricts the selection of drug molecules that can be loaded and their biomedical applications [3].
However, the microspheres are easily ruptured and destroyed by constructing large, dense holes in
small particles with diameters of less than 200 nm. In this context, the design of small microspheres with
macroporous holes that are structurally stable enough to carry large molecular drugs is an attractive
and challenging subject. Two important and rapidly developing fields that will take advantage of this
are peptide and gene therapy.
In particular, among the potential delivery platform materials, such as mesoporous materials [4–7],
hollow microspheres [8–11] and nanotubes [12,13] etc., Fe3 O4 /SiO2 hollow magnetic microspheres [14,15]
have been widely used as carriers for the storage and delivery of cargo species due to their distinctive
properties, such as facile functionalization, good biocompatibility, physical and chemical stability.
Fe3 O4 /SiO2 hollow magnetic microspheres, which are often used as a carrier, usually have a pore
structure, such as with big holes [16] or mesoporous [8,17], because of their higher loading capacity.
Materials 2017, 10, 411; doi:10.3390/ma10040411
www.mdpi.com/journal/materials
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Using magnetic hollow microspheres as carriers, cargo loading and release can be accomplished by
constructing the appropriate pore channels. Fe3 O4 /SiO2 hollow microspheres with holes that have
anisotropic properties can be called Janus particles. The methods of synthesizing Janus particles
broadly employ shielding a part of particles by embedding a monolayer of particles on a substrate
or on a Pickering emulsion [18] and then chemically modifying these particles in an aqueous phase.
In this present, we prepared the p-Fe3 O4 /SiO2 microspheres with a modified Pickering emulsion.
Additionally, the chemical composition of Fe3 O4 /SiO2 microspheres assures the efficient attachment
of organic surface functionalities such as stimuli-responsive polymer to improve the loading capacity
of cargos. The combination of a temperature sensitive polymer and Fe3 O4 /SiO2 particles can be
used as gated carriers that release cargo in response to the ambient temperature, which has potential
application in magnetic hyperthermia (42 ◦ C–49 ◦ C) [19,20]. P(NIPAM-AA) [21–23] has been widely
studied as a temperature sensitive polymer with good biocompatibility and stimulation response.
In aqueous solutions, the lower critical solution temperature (LCST) of P(NIPAM-AA) can be controlled
(32 ◦ C–49.5 ◦ C) [24]. In an aqueous, the polymer undergoes hydrophilic–hydrophobic transition
at LCST. Similarly, the cross-linked microspheres obtained by this polymer swell in water under
a critical temperature and collapse above it, and this temperature is the volume phase transition
temperature (VPTT) [25,26]. In previous studies, P(NIPAM-AA) was usually grafted onto the external
surface of inorganic microspheres [22] and the adsorption and desorption of cargos depended on the
state of the P(NIPAM-AA) network, while coating the P(NIPAM-AA) into the cavity of the hollow
microspheres can largely improve the loading capacity and reduce the release rate. However, reports
on the design and preparation of hollow microsphere with PNIPAM or P(NIPAM-AA) inside the cavity
are limited.
In this work, we combined the P(NIPAM-AA) with the hollow p-Fe3 O4 /SiO2 microspheres to
design a stimuli-responsive drug delivery system. Despite the great potential of this strategy, the use
of this composite microsphere remains largely unexplored. We developed a carrier based on bowl-like
hollow p-Fe3 O4 /SiO2 microspheres with P(NIPAM-AA) serving as a gatekeeper that allows the
encapsulation and transportation of drugs with little premature release to specific site tissue and organs
where the drug can be released upon an external alternating magnetic field. In this paper, the surface
hole size of p-Fe3 O4 /SiO2 microspheres was in the range of 18.3~37.2 nm, which allows encapsulation
of big molecules of any type. The VPTT of the P(NIPAM-AA)/Fe3 O4 /SiO2 microsphere was 42.5 ◦ C,
which meets the requirements for magnetic hyperthermia. The protein BSA was used as a model drug
to study the loading and release properties of the P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres.
2. Materials and Methods
2.1. Materials
N-isopropyl-acrylamide (NIPAM, 98%) and ammonium persulfate (APS) were purchased from
Aladdin Chemistry Co., Ltd. (Shanghai, China). N,N 0 -methylene-bis(2-propenamide) (MBA) was
purchased from Tianjin Damao Chemical Co., Ltd. (Tianjin, China). Bovine serum albumin (BSA)
was obtained from Sigma-Aldrich. Sodium acetate (CH3 COONa) and trichloromethane (CH3 Cl) were
purchased from Guangzhou Chemistry Co., Ltd. (Guangzhou, China). Ethylene glycol (EG), trisodium
citrate dehydrate (Na3 Cit) and acrylic acid (AA) were purchased from Shanghai Runjie Chemical
Reagent Co., Ltd. (Shanghai, China). Paraffin Wax (melting point 60–62 ◦ C) was purchased from
Shanghai Yonghua Paraffin Co., Ltd. (Shanghai, China). Ferric trichloride (FeCl3 ·6H2 O) was purchased
from Big Alum Chemical Reagent Factory. Tetraethoxysilane (TEOS) was obtained from China Wulian
Chemical Co., Ltd. Sodium hydroxide (NaOH) and oxalic acid (C2 H2 O4 ·2H2 O) were purchased from
Tianjin Qilun Chemical Technology Ltd. (Tianjin, China). Absolute ethyl alcohol (C2 H5 OH) was
obtained from the Tianjin Fuyu Chemical Co., Ltd. (Tianjin, China). Ammonium hydroxide (NH3 ·H2 O,
25%) was purchased from China Sun Specialty Products Co., Ltd. (Jiangsu, China).
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2.2. Preparation of the p-Fe3 O4 /SiO2 Microspheres
Synthesis of Fe3 O4 microspheres. The Fe3 O4 hollow microsphere were prepared via a solvothermal
method [27]. FeCl3 ·6H2 O (2.16 g) and 0.08 g Na3 Cit were dissolved in 64 mL of EG. Then, 5.76 g
CH3 COONa was added to the mixture until the mixture was completely dissolved. The solution was
transferred into a 100 mL Teflon-lined autoclave, which was sealed and maintained at 200 ◦ C for 15 h.
The precipitation was washed with deionized water and ethanol several times, and dried in a vacuum
oven at 60 ◦ C for 12 h.
Synthesis of Fe3 O4 /SiO2 microspheres. A layer of silica was coated on the Fe3 O4 microspheres using
a modified StÖber method [28] to form the Fe3 O4 /SiO2 microspheres. In a typical synthesis, the Fe3 O4
microspheres (100 mg) were suspended in a mixture of 20 mL ethanol, 2 mL deionized water and
0.2 mL aqueous ammonia solution. After ultrasonication for 15 min, the solution was stirred for 30 min.
Then, 30 µL of TEOS was added to the mixture and allowed to react for 10 h. The precipitant was
washed with ethanol several times and dried.
Synthesis of p-Fe3 O4 /SiO2 microspheres. The Fe3 O4 /SiO2 microspheres (50 mg) were suspended
in 20 mL of deionized water and heated to 80 ◦ C, then 300 mg of wax were added. After the
wax completely melted, the solution was stirred vigorous for 15 min, the composite particles of
wax/Fe3 O4 /SiO2 were produced and collected with a magnet. The obtained wax/Fe3 O4 /SiO2
composite particles were suspended in a 40 mL NaOH solution (1 mol/L) for 24 h to partly etch
the SiO2 . To release the Fe3 O4 /SiO2 microspheres that were partly coated with a SiO2 shell from the
wax, the produced composite particles were suspended in CHCl3 (10 mL) for 12 h at room temperature
and washed for three times to remove wax completely. The obtained microspheres were washed with
ethanol and deionized water several times. To produce the Fe3 O4 /SiO2 microspheres with surface
holes, the obtained microspheres were suspended in 20 mL of an oxalic acid solution (0.025 mol/L) for
15 min, 30 min, 45 min and 60 min. Then the p-Fe3 O4 /SiO2 microspheres were obtained after being
washed with ethanol and dried in a vacuum oven at 60 ◦ C for 12 h.
2.3. Synthesis of the P(NIPAM-AA)/Fe3 O4 /SiO2 Microspheres
p-Fe3 O4 /SiO2 (100 mg) microspheres with different pore sizes were suspended in 50 mL of
deionized water and ultrasonicated for 30 min. Then, AA was added into the suspended solution,
and the mixture was heated to 70 ◦ C under a N2 atmosphere [29]. After reacting for 4 h, NIPAM,
MBA and SDS were added sequentially and stirred for 30 min, then the APS solution was injected
slowly into the mixture and stirred for 20 h to produce the P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres.
The produced microspheres were washed with deionized water and ethanol sequentially, and dried in
a vacuum oven at 60 ◦ C for 12 h. The addition of AA, NIPAM, MBA, SDS and APS is shown in Table 1.
Table 1. Recipe for the synthesis of the P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres.
Sample 1
AA (mmol)
NIPAM (mol)
MBA (mmol)
SDS (mol)
APS (mmol)
A1
A2
A3
A4
0.44
0.88
1.32
1.76
0.06
0.12
0.18
0.24
5
10
15
20
0.007
0.014
0.021
0.028
2.63
3.96
5.27
10.52
2.4. Loading and Release
The loading and release of BSA from p-Fe3 O4 /SiO2 and P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres
was examined. The P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres (50 mg) were suspended in a 10 mL
of BSA/H2 O solution (1.5 g/L). After stirring for 24 h at 30 ◦ C, the BSA-loaded microspheres were
centrifuged, and the supernatant was used to calculate the BSA loading efficiency. The absorbance
Materials 2017, 10, 411
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of the samples was measured using a UV-Vis spectrophotometer at 280 nm. The loading efficiency is
quantified by Equation (1):
Loading efficiency mg·g−1 =
Quantity of BSA on P(NIPAM−AA)/Fe3 O4 /SiO2 (mg)
Quantity of P(NIPAM−AA)/Fe3 O4 /SiO2 (g)
(1)
The BSA-loaded microspheres (50 mg) were suspended in 10 mL of a phosphate-buffered saline
(PBS) solution at pH = 7.4 with continuous shaking. At specific time intervals, a 3-mL solution was
sampled and replaced with an equal volume of the fresh releasing medium. The BSA concentration
was measured using a UV-vis spectrometer at 280 nm, and the cumulative release rate of the BSA was
calculated from the standard curve of the BSA. The BSA release experiments were performed at 37 ◦ C
and 47 ◦ C, respectively. The cumulative release rate is quantified by Equation (2):
Cumulative release rate (%) =
Drug release capacity of P(NIPAM−AA)/Fe3 O4 /SiO2
Drug laoding capacity of P(NIPAM−AA)/Fe3 O4 /SiO2
∗ 100%
(2)
2.5. Characterizations
The crystal microstructures of the products were identified by X-ray diffraction (XRD; X’Pert
Pro, Philips, Amsterdam, The Netherlands) using Cu Kα radiation with a wavelength of 0.154 nm.
The morphologies of the products were characterized using field emission scanning electron
microscopy (Nova NanoSEM 430 m, FEI, Czech Republic, The Netherlands). TEM observation
was performed on a transmission electron microscope (CM300, Philips, Amsterdam, The Netherlands).
We use the Nano Measurer software to measure the size, porosity and surface holes of the
Fe3 O4 , Fe3 O4 /SiO2 , p-Fe3 O4 /SiO2 microspheres. More than 200 microspheres were counted and
sized to determine the average particle size, porosity, surface holes and cavity microspheres.
The Fourier transform infrared spectroscopy (FT-IR) spectra of the microspheres were measured using
a spectrometer (Vector 33-MIR, Brukev Optic, Ettlingen, Germany) ranging from 400 to 4000 cm−1
with a KBr pellet technique. The weight loss behaviors for the microspheres were examined using
thermogravimetry (TGAR5000IR, Quantachrome, FL, USA). These samples were heated from room
temperature to 600 ◦ C at 10 ◦ C/min in N2 . Differential scanning calorimetry (DSC) was conducted
using the DSC (DSC 214 polyma, Netzsch, Bavarian Asia, Germany). The samples were dispersed
in deionized water and heated at a rate of 5 ◦ C per minute and scanned in a range of 20 ◦ C to
80 ◦ C in N2 . The magnetic property of the sample was investigated on a PPMS-9 (Qyantum Design
Inc., San Diego, CA, USA) with a vibrating sample magnetometer (VSM). UV-visible spectra were
recorded using a lambda 35 Spectrometer. The BET analysis was performed on an Autosorb IQ
(Quantachrome, FL, USA). Zeta potential was measured by using SZ-100 (HORIBA, Kyoto, Japan).
Dynamic Light Scattering (DLS) measurements were carried by using nano particle analyzer SZ-100
(HORBIA, Kyoto, Japan). Suspensions of nanoparticles (0.1% w/v) were prepared in deionized water
and sonicated for 20 min before the analysis. The measurements of hydrodynamic diameter (HD) were
performed in triplicate for each sample and the mean value is reported.
3. Results and Discussion
Figure 1 shows the preparation of the P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres and their
BSA-loading and release profiles. First, the Fe3 O4 /SiO2 hollow microspheres were prepared
using a solvothermal method and a modified StÖber method. Then, Fe3 O4 /SiO2 microspheres
were partly embedded in wax to form wax/Fe3 O4 /SiO2 particles via the Pickering emulsion
method [5]. Subsequently, the unprotected SiO2 shell was etched by an aqueous solution of NaOH.
Then Fe3 O4 /SiO2 microspheres partly coated with SiO2 shell were obtained after etching wax.
When the microspheres were immersed in oxalic acid solution, the unprotected Fe3 O4 was etched
quickly, and holes were generated. Finally, microspheres with different pore sizes (represented by
p-Fe3 O4 /SiO2 ) can be obtained by controlling the oxalic acid treatment time. Before addition of
initiator (KPS), the –COOH of AA can reacted only with –OH of Fe3 O4 , which can promote the
Materials 2017, 10, 411
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synthesis of P(NIPAM-AA) into the cavity of p-Fe3 O4 /SiO2 microspheres, subsequently. Then the
P(NIPAM-AA)/Fe
Materials 2017, 10, 411 3 O4 /SiO2 microspheres were synthesized via atom transfer radical polymerization
5 of 14
of NIPAM, MBA, AA. The obtained P(NIPAM-AA)/Fe3 O4 /SiO2 composite microspheres can be used
as
drug carriers
to load biological
(BSA).
BSA
loaded
the VPTT
biological
macromolecules
(BSA). macromolecules
BSA was loaded
below
thewas
VPTT
andbelow
released
when and
the
released
when
the
temperature
was
higher
than
the
VPTT.
temperature was higher than the VPTT.
Figure
microspheres with
with surface
surface holes
holes and
and the
Figure 1.
1. Preparation
Preparationof
ofthe
theP(NIPAM-AA)/Fe
P(NIPAM-AA)/Fe33O
O44/SiO
/SiO22microspheres
the BSA
BSA
loading
and
release
schematic.
loading and release schematic.
3.1.
Microspheres
3.1. The Preparation
Preparation and
and Morphology
Morphology Characterization
Characterization of
of the
the p-Fe
p-Fe33O4/SiO22 Microspheres
Figure
Figure 22 shows
shows the
the SEM
SEM and
and TEM
TEM images
images of
of the
the microspheres
microspheres obtained
obtained during
during the
the preparation
preparation
process.
Figure
2a
shows
that
the
Fe
O
microspheres
are
spherical
and
well
dispersed,
process. Figure
the Fe33 44 microspheres
dispersed, and the
the
surface
surface of
of the
the microspheres
microspheres is
is coarse
coarse and
and has
has small
small granular
granular protrusions,
protrusions, which
which indicate
indicate that
that the
the
Fe
Fe33O4 microspheres are composed
composed of
of Fe
Fe33O44 nanoparticles [27]. The average
average particle
particle size
sizeof
ofthe
theFe
Fe33O
O44
microspheres
is
114
±
5
nm
(Size
distribution
histogram
of
Fe
O
and
the
Fe
O
/SiO
microspheres
microspheres is 114 ± 5 nm (Size distribution histogram of Fe
3 3O
4 4 and the Fe
3 3O
4 4/SiO22 microspheres
shown
Information Figure
FigureS1).
S1).After
Aftercoating
coatingthe
theSiO
SiO
the
mean
diameter
shown in Supplementary Information
2 layer,
the
mean
diameter
of
2 layer,
of
the
Fe
O
/SiO
microspheres
is
125
±
7
nm
and
the
thickness
of
the
silica
coating
was
estimated
the Fe3O34/SiO
is 125 ± 7 nm and the thickness of the silica coating was estimated to be
4 2 microspheres
2
to
be 6After
nm. the
After
the Pickering
emulsification,
the 3wax/Fe
O4 /SiO2with
particles
withindiameter
6 nm.
Pickering
emulsification,
the wax/Fe
O4/SiO2 3particles
diameter
arrange in
of
arrange
of μm
20 µm–40
µm (Supplementary
Figure
S2) were
formed
the
Fe3 O4 /SiO2
20 μm–40
(Supplementary
InformationInformation
Figure S2) were
formed
by the
Fe3O4by
/SiO
2 microspheres
microspheres
partially
in paraffin,
Fe3 O4 /SiO2were
microspheres
were homogeneously
partially embedded
in embedded
paraffin, and
Fe3O4/SiOand
2 microspheres
homogeneously
distributed on
distributed
the paraffin
(Figure
2c). Figure
2d shows
the original
uniform
the paraffinon
surface
(Figuresurface
2c). Figure
2d shows
that the
originalthat
uniform
spherical
shapespherical
becomes
shape
becomes mushroom-like
and exhibits
a Janus
structure:
the Fe3 O4 partly
microspheres
partlythe
coated
mushroom-like
and exhibits a Janus
structure:
the Fe
3O4 microspheres
coated with
SiO2
with
the
SiO
shell.
The
formation
of
the
Janus
microspheres
is
attributed
to
the
etching
of
the
SiO
shell
shell. The formation
of the Janus microspheres is attributed to the etching of the SiO2 shell layer
by
2
2
layer
by The
NaOH.
p-Fe23microspheres
O4 /SiO2 microspheres
were obtained
afterfor
etching
for(Figure
15 min (Figure
2e),
NaOH.
p-FeThe
3O4/SiO
were obtained
after etching
15 min
2e), 30 min
30
min (Figure
min (Figure
2g),the
andporosity
the porosity
was 47%,
respectively;
(Figure
2f) and2f)
45and
min45(Figure
2g), and
was 47%,
59%,59%,
and and
76%,76%,
respectively;
the
the
corresponding
average
surface
holes
diameter
wasapproximately
approximately20.3
20.3±±5 5nm,
nm, 28.3
28.3 ±± 88 nm and
corresponding
average
surface
holes
diameter
was
and
37.2
37.2 ±
± 7 nm. The porosity, the pore size
size and
and the
the number
number of
of surface
surface holes
holes increase
increase with
with increasing
increasing
etching
etching time.
time. However, most of the microspheres were broken when the etching
etching time
time was
was 60
60 min
min
(Figure
Figure2i2ishows
showsthat
thatmost
mostofofthe
themicrospheres
microsphereshave
have
a hollow
structure.
of
(Figure 2h). Figure
a hollow
structure.
TheThe
sizesize
of the
the
is approximately
7 nm,
and
averagesize
sizeofof the
the cavity is
Fe3OFe
4/SiO
2 microspheres
is approximately
125 125
± 7±nm,
and
thetheaverage
is
3 O4 /SiO
2 microspheres
approximately
60
nm.
approximately 60 nm.
Materials 2017, 10, 411
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Figure 2. The SEM and TEM images of the microspheres obtained during the preparation process.
Figure 2. The SEM and TEM images of the microspheres obtained during the preparation process.
(a) Fe3O4; (b) Fe3O4/SiO2; (c) wax/Fe3O4/SiO2 (d) Fe3O4 microspheres partly coated with a SiO2 shell;
(a) Fe3 O4 ; (b) Fe3 O4 /SiO2 ; (c) wax/Fe3 O4 /SiO2 (d) Fe3 O4 microspheres partly coated with a SiO2 shell;
(e–h) p-Fe3O4/SiO2 after oxalic acid corrosion for different times ((e) 15 min; (f) 30 min; (g) 45 min;
(e–h) p-Fe3 O4 /SiO2 after oxalic acid corrosion for different times ((e) 15 min; (f) 30 min; (g) 45 min;
(h) 60 min) (i) TEM image of the Fe3O4/SiO2 microspheres.
(h) 60 min) (i) TEM image of the Fe3 O4 /SiO2 microspheres.
3.2. Porous Structure Characterization of the p-Fe3O4/SiO2 Microspheres
3.2. Porous Structure Characterization of the p-Fe3 O4 /SiO2 Microspheres
To study the pore structure of the p-Fe3O4/SiO2 microspheres, N2 adsorption/desorption experiments
To study the pore structure of the p-Fe3 O4 /SiO2 microspheres, N2 adsorption/desorption
were performed. Figure 3a shows the N2 adsorption/desorption
isotherms for the Fe3O4/SiO2
experiments were performed. Figure 3a shows the N2 adsorption/desorption isotherms for the
microspheres and p-Fe3O4/SiO2 microspheres etched with oxalic acid with different times. All the
Fe3 O4 /SiO2 microspheres and p-Fe3 O4 /SiO2 microspheres etched with oxalic acid with different times.
isotherms
are type IV adsorption isotherms, as defined by IUPAC [30]. The BET surface area of the
All the isotherms are type IV adsorption isotherms, as defined
by IUPAC [30]. The BET surface
Fe3O4/SiO2 microspheres without oxalic acid etching was 32.9 m2/g. After etching2 for 15 min, 30 min
area of the Fe3 O4 /SiO2 microspheres without oxalic acid etching was 32.9 m /g.
After etching
and 45 min, the BET surface areas of the p-Fe3O4/SiO2 microspheres were 38.3 m2/g, 42.2 m2/g and
for 15 min, 30 min and 45 min, the BET surface areas of the p-Fe3 O4 /SiO2 microspheres were
39.6 m22/g, respectively,
which shows an increase compared to the un-etched microspheres. However,
38.3 m /g, 42.2 m2 /g and 39.6 m2 /g, respectively, which shows an increase compared to the un-etched
the surface areas of the microspheres prepared at various etching times are similar. We suspect this
microspheres. However, the surface areas of the microspheres prepared at various etching times are
is because the surface holes are enlarged, which decreases the specific surface area as the oxalic acid
similar. We suspect this is because the surface holes are enlarged, which decreases the specific surface
continues to etch. However, the formation of new pores increase the surface area. For these two
area as the oxalic acid continues to etch. However, the formation of new pores increase the surface area.
aspects, the surface area of the microspheres see little change with increased etching time.
For these two aspects, the surface area of the microspheres see little change with increased etching time.
Materials 2017, 10, 411
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Figure 3.
3. The
The adsorption/desorption
adsorption/desorption isotherms
the corresponding
corresponding BJH
BJH pore
pore size
size distribution
distribution
Figure
isotherms (a)
(a) and
and the
O44/SiO
4/SiO2 microspheres (b,c).
for the
the Fe
Fe33O
for
/SiO2 2and
andp-Fe
p-Fe3O
3 O4 /SiO2 microspheres (b,c).
The pore size distribution of microspheres mainly ranged from 10 nm–100 nm (Figure 3b). The
The pore size distribution of microspheres mainly ranged from 10 nm–100 nm (Figure 3b).
pore size that appeared near 60 nm could be associated with microsphere cavities, which agrees with
The pore size that appeared near 60 nm could be associated with microsphere cavities, which agrees
the TEM images. In addition, the p-Fe3O4/SiO2 microsphere at 30 min and 45 min had a pore size
with the TEM images. In addition, the p-Fe3 O4 /SiO2 microsphere at 30 min and 45 min had a pore size
distribution from 4 nm to 10 nm (Figure 3c). It is obvious to find that the surface areas of microspheres
distribution from 4 nm to 10 nm (Figure 3c). It is obvious to find that the surface areas of microspheres
at 30 min and 45 min are similar, but the porosity at 45 min is higher. The higher the porosity,
at 30 min and 45 min are similar, but the porosity at 45 min is higher. The higher the porosity, the higher
the higher the loading capacity, which is consistent with our following experimental results. Thus,
the loading capacity, which is consistent with our following experimental results. Thus, we focus on
we focus on the study of p-Fe3O4/SiO2 microspheres which were etched for 45 min by oxalic acid
the study of p-Fe3 O4 /SiO2 microspheres which were etched for 45 min by oxalic acid grafting with
grafting with P(NIPAM-AA).
P(NIPAM-AA).
3.3. Morphology
Morphology Characterization
Characterization of
of the
the P(NIPAM-AA)/Fe
P(NIPAM-AA)/Fe3O
O4/SiO2 Microspheres
3.3.
3 4 /SiO2 Microspheres
The SEM
P(NIPAM-AA)/Fe
3O4/SiO2 microspheres obtained (under different amount
The
SEM images
imagesofofthe
the
P(NIPAM-AA)/Fe
3 O4 /SiO2 microspheres obtained (under different
of
monomer
addition
shown
in
Table
1)
are
shown
TheP(NIPAM-AA)/Fe
P(NIPAM-AA)/FeO3O/SiO
4/SiO2
amount of monomer addition shown in Table 1) are
showninin Figure
Figure 4.4.The
3 4
2
microspheres
were
produced
via
atom
transfer
radical
polymerization
of
NIPAM,
MBA
and
AA,
microspheres were produced via atom transfer radical polymerization of NIPAM, MBA and AA,
after the
the esterification
3O4/SiO2 (etching for 45 min) microspheres. It is
after
esterification reaction
reactionbetween
betweenAA
AAand
andp-Fe
p-Fe
3 O4 /SiO2 (etching for 45 min) microspheres.
interesting
that
some
small
protrusions
appeared
in
the
(marked
withwith
arrows
in Figure
4b). 4b).
We
It is interesting that some small protrusions appeared in holes
the holes
(marked
arrows
in Figure
suspect
that this
P(NIPAM-AA)
was grafted
the holes
p-Fe
3O4/SiO2 microspheres.
We
suspect
thatwas
thisbecause
was because
P(NIPAM-AA)
wasinto
grafted
intoofthe
holes
of p-Fe3 O4 /SiO2
The
size
of
microspheres
increased
with
increasing
amounts
of
monomers,
the
HD of p-Fe
4/SiO2
microspheres. The size of microspheres increased with increasing amounts of monomers,
the3OHD
of
microspheres
was
102
±
9
nm,
and
after
grafting
P(NIPAM-AA),
the
corresponding
values
were
p-Fe3 O4 /SiO2 microspheres was 102 ± 9 nm, and after grafting P(NIPAM-AA), the corresponding
120 ± 17
nm 120
(sample
167 ±A1)
21 nm
A2)
(Supplementary
Information Figure
S3). The
values
were
± 17A1)
nmand
(sample
and(sample
167 ± 21
nm
(sample A2) (Supplementary
Information
HD
distribution
curves
also
show
the
good
dispersibility
of
samples
A1
and
A2.
From
Figure
4b–d,
Figure S3). The HD distribution curves also show the good dispersibility of samples A1 and
A2.
with
the
increasing
amount
of
monomers,
the
holes
gradually
disappeared,
and
big
aggregated
From Figure 4b–d, with the increasing amount of monomers, the holes gradually disappeared, and big
particles also
appeared
circles
in circles
Figure in
4c,d),
which
be proved
by HD by
results
aggregated
particles
also(marked
appearedwith
(marked
with
Figure
4c,d),can
which
can be proved
HD
(Supplementary
Information
Figure
S3)
[31].
results (Supplementary Information Figure S3) [31].
Materials 2017, 10, 411
Materials 2017, 10, 411
Materials 2017, 10, 411
8 of 14
8 of 14
8 of 14
Figure 4. SEM images and HD distribution curves of the P(NIPAM-AA)/Fe3O4/SiO2 microspheres
Figure 4. SEM images and HD distribution curves of the P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres
prepared
with
variousand
amounts
of NIPAM, MBA,
AA.
A1; (b) sample3OA2,
(c) sample A3
Figure
4. SEM
images
HD distribution
curves
of(a)
theSample
P(NIPAM-AA)/Fe
4/SiO2 microspheres
prepared
various
andwith
(d) sample
A4.amounts of NIPAM, MBA, AA. (a) Sample A1; (b) sample A2;, (c) sample A3
prepared with various amounts of NIPAM, MBA, AA. (a) Sample A1; (b) sample A2, (c) sample A3
and (d) sample A4.
and (d) sample A4.
3.4. Characterization of the P(NIPAM-AA)/Fe3O4/SiO2 Microspheres
3.4.3.4.
Characterization
of ofthetheP(NIPAM-AA)/Fe
Microspheres
3O
44/SiO
22 Microspheres
Figure 5 presents
the
FTIR spectra of
Fe
3/SiO
O4, p-Fe
3O4/SiO2, P(NIPAM-AA) and P(NIPAM-AA)/
Characterization
P(NIPAM-AA)/Fe
3O
Fe3O4/SiO2 microspheres. The FTIR spectrum of Fe3O4 microspheres showed the peak at 567 cm−1 is
Figure
5 presents
thethe
FTIR
spectra
ofof
FeFe
P(NIPAM-AA)and
andP(NIPAM-AA)/
P(NIPAM-AA)/
3O
4 ,4,p-Fe
Figure
5 to
presents
FTIR
spectra
3O
p-Fe3 3O
O44/SiO
/SiO2,2 ,P(NIPAM-AA)
attributed
the stretching
Fe-O
of Fe3O
4 and at 1622 cm−1 and 1389 cm−1 is attributed to the anti−1
Fe3Fe
O34O
/SiO
Thevibration
FTIR spectrum
spectrum
ofFe
Fe3O
O
showed
peak
at 567
2 2microspheres.
3indicate
4 microspheres
4/SiO
microspheres.
The
FTIR
4 microspheres
showed
thethe
peak
at 567
cm−1cm
is
symmetrical
and symmetric
of COO−of
which
the existence
of carboxylate
groups
on
−
1
−
1
is attributed
to
the stretching
stretching
Fe–O
at 1622
and 1389
cm
is attributed
to the
4 and
attributed
the
Fe-O
ofof
FeFe
3O34O
and
at
cm−1−1cm
1389
cm
attributed
to theofantithe Fe3Oto
4 microspheres.
The
characteristic
band
at 1622
1097
cm
isand
attributed
to −1
theis
Si–O–Si
vibration
−
−
anti-symmetrical
and
symmetric
vibration
of COO
which
theofexistence
of carboxylate
the FTIR and
spectrum
of Fe3O
4/SiO
2 microspheres.
The
FTIRindicate
spectrum
P(NIPAM-AA)/Fe
3Ogroups
4/SiO2groups
symmetrical
symmetric
vibration
of COO
which
indicate
the existence
of carboxylate
on
−1 is attributed
−1
−1
−1 is
microspheres
showedThe
the
characteristic
peaks
1537
(stretching
peak
of
1650
cm
on the
the Fe
Fe33OO4 4microspheres.
microspheres.
The
characteristic
band
1097
toN–H),
the Si–O–Si
vibration
characteristic
band
at at
1097
cmcm
attributed
to the
Si–O–Si
vibration
of
(stretching
peak of
and
1394
cm−1 (C-N stretching
peak),spectrum
which confirmed
that P(NIPAM-AA)
of the
ofofC=O)
FeFe
O
/SiO
microspheres.
The
FTIR
of
P(NIPAM-AA)/Fe
O
/SiO
the FTIR
FTIR spectrum
spectrum
3
O
4
/SiO
2
microspheres.
The
FTIR
P(NIPAM-AA)/Fe
3
O
4
/SiO
2 2
3 4
2
3 4
was
successfully
grafted
to
the
p-Fe
3O4/SiO2 microspheres. −
1
−1
−1
microspheres
showedthe
thecharacteristic
characteristicpeaks
peaksatat1537
1537cm
cm (stretching
microspheres
showed
(stretching peak
peakof
of N–H),
N–H),1650
1650cm
cm−1
−1
−
1
(stretching
peak
C=O)
and1394
1394cm
cm (C–N
(C-N stretching
that
P(NIPAM-AA)
(stretching
peak
of of
C=O)
and
stretchingpeak),
peak),which
whichconfirmed
confirmed
that
P(NIPAM-AA)
was
successfully
grafted
to
the
p-Fe
3
O
4
/SiO
2
microspheres.
was successfully grafted to the p-Fe3 O4 /SiO2 microspheres.
Figure 5. Infrared spectra of (a) Fe3O4; (b) Fe3O4/SiO2; (c) P(NIPAM-AA) and (d) P(NIPAM-AA)/Fe3O4/SiO2
microspheres.
The organic compound content in the composite microspheres were determined using TGA
Figure
Infrared
spectra
FeFe
3O4;O
(b) Fe
3OFe
4/SiO
2; (c)
P(NIPAM-AA)
and (d)200
P(NIPAM-AA)/Fe
3O4/SiO
measurements,
asspectra
shownofof
in(a)
Figure
cases,
the
loss below
°C (d)
can P(NIPAM-AA)/
be ascribed
to2
Figure
5. 5.Infrared
(a)
(b)all
; (c) P(NIPAM-AA)
and
3 6.4 ;In
3 O4 /SiO
2weight
microspheres.
desorption
of
the
physically
bonded
small
molecule
monomers
and
water.
The
weight
loss
in
the
Fe3 O4 /SiO2 microspheres.
200 °C–450 °C temperature range can be attributed to the decomposition of the polymer, and the
The organic compound content in the composite microspheres were determined using TGA
The organic compound
microspheres
were
using TGA
measurements,
as shown in content
Figure 6.inInthe
all composite
cases, the weight
loss below
200determined
°C can be ascribed
to
◦
measurements,
as
shown
in
Figure
6.
In
all
cases,
the
weight
loss
below
200
C
can
be
ascribed
desorption of the physically bonded small molecule monomers and water. The weight loss in the to
desorption
of °C
thetemperature
physically bonded
small
molecule to
monomers
and water.
Thepolymer,
weight loss
200 °C–450
range can
be attributed
the decomposition
of the
and in
thethe
200 ◦ C–450 ◦ C temperature range can be attributed to the decomposition of the polymer, and the
Materials 2017, 10, 411
9 of 14
Materials 2017, 10, 411
9 of 14
residual
weight
can
loss of
Materials
2017, 10,
411be ascribed to Fe3 O4 and SiO2 . For samples A1, A2, A3 and A4, the weight
9 of 14
residual
weight
can be ascribed to Fe3O4 and SiO2. For samples A1, A2, A3 and A4, the weight loss
of
the polymer was 5.33%, 13.18%, 26.03% and 33.28%, respectively, and the corresponding weight ratio
the polymer was 5.33%, 13.18%, 26.03% and 33.28%, respectively, and the corresponding weight ratio
residual weight can of
be ascribed
to Fe2.83,
3O4 and SiO
2. For samples
A1,the
A2,proportion
A3 and A4, of
the weight
loss ofin the
of inorganic/polymer
6.26,
that
polymer
of inorganic/polymer 17.76,
of 17.76,
6.26, 2.83,2.00,
2.00, suggesting
suggesting that
the proportion
of thethe
polymer
in the
the
polymer
was
5.33%,
13.18%,
26.03%
and
33.28%,
respectively,
and
the
corresponding
weight
ratio
composite
microspheres
increased
with
increasing
of monomer.
monomer.
composite
microspheres
increased
with
increasingamounts
amounts of
of inorganic/polymer of 17.76, 6.26, 2.83, 2.00, suggesting that the proportion of the polymer in the
composite microspheres increased with increasing amounts of monomer.
6. TGA
curves
of the
3O4/SiO2 microspheres and P(NIPAM-AA)/Fe3O4/SiO2 microspheres.
FigureFigure
6. TGA
curves
of the
p-Fep-Fe
3 O4 /SiO2 microspheres and P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres.
Figure 6. TGA curves of the p-Fe3O4/SiO2 microspheres and P(NIPAM-AA)/Fe3O4/SiO2 microspheres.
The room temperature magnetization hysteresis curve of the p-Fe3O4/SiO2 microspheres (Figure 7)
The
room
temperature
magnetization
hysteresis
of theare
p-Fe
3 O4 /SiO2 microspheres
exhibits
negligible
hysteresis,
which indicates
that the curve
microspheres
superparamagnetic.
For
The
room
temperature
magnetization
hysteresis
curve
of
the
p-Fe
3O4/SiO2 microspheres (Figure 7)
(Figure
7) exhibits
negligible
hysteresis,
which
indicates
that the microspheres
are superparamagnetic.
samples
A1–A3,
the magnetization
curves
of the
P(NIPAM-AA)/Fe
3O4/SiO2 microspheres was similar
exhibits negligible
hysteresis, which indicates
the
microspheres are O
superparamagnetic.
For
For samples
curves which
ofthat
theindicated
P(NIPAM-AA)/Fe
to that ofA1–A3,
the barethe
p-Femagnetization
3O4/SiO2 microspheres,
that the grafting
polymer
can preserve was
3 4 /SiO
2 microspheres
samples A1–A3, the magnetization curves of the P(NIPAM-AA)/Fe3O4/SiO2 microspheres was similar
similar
to that
of the bare p-Fe
indicated
thatthe
theP(NIPAM-AA)/
grafting polymer
their
superparamagnetic
properties.
For
sample A4, thewhich
magnetization
curve
3 O4 /SiO
2 microspheres,
to that of the bare p-Fe3O4/SiO2 microspheres, which indicated that the grafting polymer can preserve
Fe3O4/SiOtheir
2 microspheres
suggest the microspheres
and we suspected
can preserve
superparamagnetic
properties. was
Fornon-superparamagnetic
sample A4, the magnetization
curve the
their superparamagnetic properties. For sample A4, the magnetization curve the P(NIPAM-AA)/
that was due to
formation
of
Fe
2O3 via the
partial
oxidation
of
Fe
3O4. Fe
2O3 non-superparamagnetic
is
superparamagnetic
P(NIPAM-AA)/Fe
O
/SiO
microspheres
suggest
the
microspheres
was
3 4
2
Fe3O4/SiO2 microspheres
suggest
the microspheres was non-superparamagnetic and we suspected
when the particle
size
is smaller
20 nm [32–34].
However, thepartial
diameter of Fe3O4 is
nm,
and we
was
due
to2than
of Feoxidation
ofabout
Fe3 O114
2 O3 via the
4 . Fe2 O3 is
thatsuspected
was due tothat
formation
of Fe
Oformation
3 via the partial
of Fe3O4. Feoxidation
2O3 is superparamagnetic
so the crystal structures of the p-Fe3O4/SiO2 microspheres were analyzed via XRD (Supplementary
superparamagnetic
the particle
size
smaller
than 20the
nmdiameter
[32–34].ofHowever,
the114
diameter
of
when the particlewhen
size is smaller
than 20
nmis[32–34].
However,
Fe3O4 is about
nm,
Information Figure S4). All the diffraction peaks agreed with the magnetic cubic structure of Fe3O4
the
crystal
structures
of
the
p-Fe
3
O
4
/SiO
2
microspheres
were
analyzed
via
XRD
(Supplementary
Fe3 Oso
is
about
114
nm,
so
the
crystal
structures
of
the
p-Fe
O
/SiO
microspheres
were
analyzed
via
4
4
2
(JCPDS No. 19-0629). The grain size of the prepared Fe3O43 microspheres
calculated by the Scherrer
FigureInformation
S4). All the diffraction
peaks
agreed
with thepeaks
magnetic
cubic
structure
of Fe3O4
XRD Information
(Supplementary
Figure
S4).
All
the
diffraction
agreed
with
theamagnetic
formula at the (311) plane was 20.8 nm, which indicated that the microspheres
had
secondarycubic
(JCPDS
No.
19-0629).
The
grain
size
of
the
prepared
Fe
3O4 microspheres calculated by the Scherrer
structure
of Fe[27].
3 O4 (JCPDS No. 19-0629). The grain size of the prepared Fe3 O4 microspheres calculated
structure
formula
at the
(311) plane
20.8
nm, was
which
indicated
that the
microspheres
hadmicrospheres
a secondary had
by the
Scherrer
formula
at thewas
(311)
plane
20.8
nm, which
indicated
that the
structure [27].
a secondary structure [27].
Figure 7. The VSM curves for the p-Fe3O4/SiO2 microspheres and P(NIPAM-AA)/Fe3O4/SiO2 composite
microspheres prepared under various conditions.
Figure 7. The VSM curves for the p-Fe3O4/SiO2 microspheres and P(NIPAM-AA)/Fe3O4/SiO2 composite
Figure
7. The VSM curves for the p-Fe3 O4 /SiO2 microspheres and P(NIPAM-AA)/Fe3 O4 /SiO2
microspheres prepared under various conditions.
The saturation
magnetization
(Ms) values
the p-Fe3O4/SiO2 microspheres and P(NIPAM-AA)/
composite
microspheres
prepared under
variousofconditions.
Fe3O4/SiO2 composite microspheres A1, A2, A3, and A4 were 78.2, 75.8, 72.7, 35 and 6 emu/g,
The saturation magnetization (Ms) values of the p-Fe3O4/SiO2 microspheres and P(NIPAM-AA)/
respectively.
The
Ms values of the
3O4/SiO2 microspheres decreased rapidly with
The
magnetization
(MsP(NIPAM-AA)/Fe
)A1,
values
p-Fe
4 /SiO
2 microspheres
Fe3O4saturation
/SiO2 composite
microspheres
A2, of
A3,the
and
A43 Owere
78.2,
75.8, 72.7, 35and
andP(NIPAM-AA)/
6 emu/g,
the increase of the proportion of polymer, which was consistent with the TGA results. The prepared
Fe3 Orespectively.
/SiO
composite
microspheres
A1,
A2,
A3,
and
A4
were
78.2,
75.8,
72.7,
and 6
emu/g,
The Ms values of the P(NIPAM-AA)/Fe3O4/SiO2 microspheres decreased 35
rapidly
with
4
2
P(NIPAM-AA)/Fe3O4/SiO2 microspheres could be easily separated using a magnet (inset photographs),
the increase
of the
polymer,
which was consistent
with
the TGA results.decreased
The prepared
respectively.
The
Msproportion
values ofofthe
P(NIPAM-AA)/Fe
rapidly
3 O4 /SiO
2 microspheres
which demonstrated
their strong magnetic response.
4/SiOproportion
2 microspheres
be easilywhich
separated
using
a magnet with
(inset photographs),
with P(NIPAM-AA)/Fe
the increase of3Othe
ofcould
polymer,
was
consistent
the TGA results.
which demonstrated
their strongO
magnetic
The prepared
P(NIPAM-AA)/Fe
/SiO response.
microspheres could be easily separated using a magnet
3
4
2
(inset photographs), which demonstrated their strong magnetic response.
Materials 2017, 10, 411
Materials 2017, 10, 411
10 of 14
10 of 14
The microspheres used as carrier should have good dispersibility. Also, the higher of the content
The2017,
microspheres
as carriercapacity.
should have
good dispersibility.
Also,
theused
higher
content
of polymer,
the
higher
of used
the loading
Therefore,
sample A2
was
asofa the
carrier
Materials
10,
411
10 offor
14 the
of
polymer,
the
higher
of
the
loading
capacity.
Therefore,
sample
A2
was
used
as
a
carrier
for
the
subsequent loading and release of the protein.
The microspheres
astemperature
carrier
have good
Also,important
the higher of
the content
subsequent
loadingtransition
andused
release
of the should
protein.(VPTT)
A
volume
phase
is dispersibility.
one of the most
property
for the
of polymer,
thephase
highertransition
of the loading
capacity.
Therefore,
A2 was
used as property
a carrier for the
A volume
temperature
(VPTT)
is onesample
of the most
important
cross-linked composite particles obtained from the thermosensitive polymer [26,35,36]. Therefore,
subsequent loading
andparticles
release of
the protein.
cross-linked
composite
obtained
from the thermosensitive polymer [26,35,36]. Therefore, the
the VPTT of sample A2 microspheres was determined by using DSC measurements. The DSC curves
transition temperature
(VPTT)
one DSC
of themeasurements.
most important
property
for the
VPTTAofvolume
samplephase
A2 microspheres
was determined
by is
using
The
DSC curves
of
of thethe
P(NIPAM-AA)/Fe
/SiO2 2microspheres
microspheres
(Figure
8) show
peak
onset
temperature
33O
44/SiO
cross-linked
composite
particles
obtained
from(Figure
the
thermosensitive
polymer
[26,35,36].
Therefore,
the was
P(NIPAM-AA)/Fe
O
8) show
thatthat
the the
peak
onset
temperature
was
◦ C (heating curve) and 49.5 ◦ C (cooling curve), which are the transition
observed
atofapproximately
42.542.5
VPTT
sample
A2 microspheres
was determined
by 49.5
using
measurements.
The
curves of
observed
at approximately
°C (heating
curve) and
°CDSC
(cooling
curve), which
areDSC
the transition
temperatures
(VPTT)
[25,26].
the P(NIPAM-AA)/Fe
3O4/SiO2 microspheres (Figure 8) show that the peak onset temperature was
temperatures
(VPTT)
[25,26].
observed at approximately 42.5 °C (heating curve) and 49.5 °C (cooling curve), which are the transition
temperatures (VPTT) [25,26].
8. DSC
curve
of the
P(NIPAM-AA)/Fe33O
O44/SiO
microspheres
(Sample
A2). A2).
FigureFigure
8. DSC
curve
of the
P(NIPAM-AA)/Fe
/SiO2 2composite
composite
microspheres
(Sample
P(NIPAM-AA)
chains
also
be absorbed onto
the2surface
of the
p-Fe3O4/SiO
2 as demonstrated
Figure 8. DSC
curve can
of the
P(NIPAM-AA)/Fe
3O4/SiO
composite
microspheres
(Sample
A2).
P(NIPAM-AA)
also be absorbed
onto(HD)
the surface
the p-Fe
3 O4 /SiO2 as demonstrated
by the change chains
in the can
hydrodynamic
diameter
of the ofhybrid
P(NIPAM-AA)/Fe
3O4/SiO2
by the
change
inobserved
the hydrodynamic
(HD)
the of
hybrid
P(NIPAM-AA)/Fe
3 Othe
4 /SiO2
P(NIPAM-AA)
chains
be diameter
absorbed onto
the9of
surface
the
p-Fe
3O4/SiO
2 as demonstrated
microspheres
by can
DLSalso
measurements.
Figure
shows
the
temperature
dependence
of
microspheres
observed
by hydrodynamic
DLS measurements.
Figure
9 shows
the 162
temperature
of
by the
change
the
diameter
(HD)
of
the hybrid
P(NIPAM-AA)/Fe
3O4/SiO2
size
of the
hybridin
microspheres.
The average
HD that
was approximately
nm at 28 °C,dependence
decreasing
microspheres
Figure
9 shows
theintemperature
dependence
ofatthe
to
135
nm hybrid
at observed
54 °C.
Atby28DLS
°C measurements.
the polymer
chains
are
present
their full extended
hydrated
the size
of the
microspheres.
The average
HD
that
was
approximately
162 nm
28 ◦ C,
◦ Caverage
size ofto
the135
hybrid
microspheres.
HD
that
wasWhen
approximately
nmfull
at 28above
°C, decreasing
conformation
thusat
increasing
overall
of
particles.
the was temperature
the VPTT
decreasing
nm
54 ◦ C. Atthe
28The
theHD
polymer
chains
are present
in162
their
extended
hydrated
to
135
nm
at
54
°C.
At
28
°C
the
polymer
chains
are
present
in
their
full
extended
hydrated
of
the
P(NIPAM-AA),
the
hydrated
bonding
between
NIPAM,
AA
units
and
water
molecules
is the
conformation thus increasing the overall HD of particles. When the was temperature
above
conformation
thus
increasing
the
overall
HD of particles.
When
the was temperature
above
the of
VPTT
disrupted,
and
the
extended
chains
of
polymer
shrink
in
the
globular
form.
Hence,
the
HD
the
VPTT of the P(NIPAM-AA), the hydrated bonding between NIPAM, AA units and water molecules
of the P(NIPAM-AA),
thetemperature.
hydrated bonding
between
NIPAM,
units and water
molecules
is
particles
decreases at this
These results
confirm
the AA
thermoresponsive
behavior
of the
is disrupted, and the extended chains of polymer shrink in the globular form. Hence, the HD of the
disrupted,
and
the
extended
chains
of
polymer
shrink
in
the
globular
form.
Hence,
the
HD
of
the
grafted polymer and also prove the good dispersibility of nanoparticles [37,38]. The zeta potential
particles
decreases
at thisthis
temperature. These
results
confirm
thethermoresponsive
thermoresponsive behavior
of the
particles
results
confirm
the
value
for decreases
the p-Fe3Oat
4/SiO2 temperature.
at pH 6.8 wasThese
−21.4 mV,
which
uponthe
polymer grafting wasbehavior
−16.8 mV.ofThis
grafted
polymer
and
alsoalso
prove
thethe
good
of nanoparticles
nanoparticles
[37,38].
zeta
potential
grafted
polymer
and
prove
gooddispersibility
dispersibility
of
TheThe
zeta
potential
change
of zeta
potential
can
be
attributed
to the fact that
some of the [37,38].
free
hydroxyl
groups
are
valueconsumed
for
the
p-Fe
O
/SiO
at
pH
6.8
was
−
21.4
mV,
which
upon
polymer
grafting
was
−
16.8 mV.
3 43polymer
2 2 at
value for the
O4/SiO
pH 6.8 was −21.4 mV, which upon polymer grafting was −16.8 mV. This
byp-Fe
the
[39,40].
This change
of zeta
zeta potential
potentialcan
canbebeattributed
attributed
fact
that
some
of the
hydroxyl
groups
change of
to to
thethe
fact
that
some
of the
freefree
hydroxyl
groups
are are
consumed
by polymer
the polymer
[39,40].
consumed
by the
[39,40].
Figure 9. The effect of temperature on the hydrodynamic diameter of P(NIPAM-AA)/Fe3O4/SiO2
microspheres of sample 2.
Figure
9. effect
The effect
of temperature
hydrodynamicdiameter
diameter of
of P(NIPAM-AA)/Fe
P(NIPAM-AA)/Fe3O4/SiO
2
Figure
9. The
of temperature
on on
thethe
hydrodynamic
3 O4 /SiO2
microspheres of sample 2.
microspheres of sample 2.
Materials 2017, 10, 411
11 of 14
Materials 2017, 10, 411
11 of 14
3.5. BSA Loading and Release
3.5. BSA Loading and Release
P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres and p-Fe3 O4 /SiO2 microspheres were used for
P(NIPAM-AA)/Fe3O4/SiO2 microspheres and p-Fe3O4/SiO2 microspheres were used for the
the adsorption of BSA at different temperatures (Table 2). Table 2 shows that the BSA loading
adsorption of BSA at different temperatures (Table 2). Table 2 shows that the BSA loading capacity
capacity increased with the increasing etching time of oxalic acid and the P(NIPAM-AA) grafting.
increased with the increasing etching time of oxalic acid and the P(NIPAM-AA) grafting. the
The corresponding BSA loading capacity of p-Fe3 O4 /SiO2 microspheres increased by 31.0%, 71.7%
corresponding BSA loading capacity of p-Fe3O4/SiO2 microspheres increased by 31.0%, 71.7% and
and 84.8%, respectively, but the specific surface area of the microspheres increased by 16.4%, 28.6%
84.8%, respectively, but the specific surface area of the microspheres increased by 16.4%, 28.6% and
and 20.4%, respectively, after 15 min, 30 min and 45 min of oxalic acid etching. This may be related to
20.4%, respectively, after 15 min, 30 min and 45 min of oxalic acid etching. This may be related to the
the formation of large holes and the mesoporous pores caused by the oxalic acid etching, and BSA
formation of large holes and the mesoporous pores caused by the oxalic acid etching, and BSA
(3.5 nm [41]) can only enter the interior of the microsphere through surface holes that are large enough
(3.5 nm [41]) can only enter the interior of the microsphere through surface holes that are large
and adsorb on the surface. The loading of BSA increased with increases in the porosity, holes and
enough and adsorb on the surface. The loading of BSA increased with increases in the porosity, holes
mesoporous ratio. The BSA loading capacity was further enhanced after grafting P(NIPAM-AA)
and mesoporous ratio. The BSA loading capacity was further enhanced after grafting P(NIPAM-AA)
because BSAs are absorbed in the P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres through hydrogen
because BSAs are absorbed in the P(NIPAM-AA)/Fe3O4/SiO2 microspheres through hydrogen
bonding and van der wale forces between carboxyl-carboxyl groups. This is because the carbonyl
bonding and van der wale forces between carboxyl-carboxyl groups. This is because the carbonyl
groups of P(NIPAM-AA) can form hydrogen bonds with the amino groups and carbonyl groups of
groups of P(NIPAM-AA) can form hydrogen bonds with the amino groups and carbonyl groups of
BSA (proved by FTIR in Supplementary Information Figure S5).
BSA (proved by FTIR in Supplementary Information Figure S5).
Table 2. The BSA loading capacity of the microspheres at 37 ◦ C and 47 ◦ C.
Table 2. The BSA loading capacity of the microspheres at 37 °C and 47 °C.
Microspheres
Corrosion
Microspheres Corrosion
Time
(min)
Time (min)
0
15
15
30
30
45
45
Loading Capacity
(mg/g)
Loading
Capacity
(mg/g)
P(NIPAM-AA)/Fe
4/SiO
P(NIPAM-AA)/Fe
/SiO
3 O34O
2 2
p-Fe
p-Fe33O
O44/SiO
/SiO22Microspheres
Microspheres
Microspheres
Microspheres
18.4
31.6
18.4
31.6
24.1
41.8
24.1
41.8
31.6
55.2
31.6
55.2
34.0
74.0
34.0
74.0
◦ C; thus, the release behavior of the BSA-loaded microspheres
The
VPTT of
P(NIPAM-AA) is
The VPTT
of P(NIPAM-AA)
is 42.5
42.5 °C;
thus, the release behavior of the BSA-loaded microspheres
◦
◦ C (below the VPTT). Figure 10 shows that reaching
was
C (above
was studied
studied at
at 47
47 °C
(above the
the VPTT)
VPTT) and
and 37
37 °C
(below the VPTT). Figure 10 shows that reaching
the
state required
required lesser
the equilibrium
equilibrium state
lesser time
time and
and the
the cumulative
cumulative release
release rate
rate was
was higher
higher when
when the
the
◦ C to 47 ◦ C. Also, the cumulative release rate of BSA loaded on the
temperature
changed
from
37
temperature changed from 37 °C to 47 °C. Also, the cumulative release rate of BSA loaded on the
P(NIPAM-AA)/Fe
O44/SiO
/SiO
withholes
holeswas
waslower
lowerthan
thanthat
thatwithout
withoutholes
holes(Figure
(Figure10a).
10a).
2 microspheres
P(NIPAM-AA)/Fe33O
2 microspheres
with
ss
Figure 10.
10. The
3O4/SiO2 microspheres at 37 °C ◦
(a)
Figure
The release
release curves
curves of
of the
the BSA
BSA loaded
loaded P(NIPAM-AA)/Fe
P(NIPAM-AA)/Fe
3 O4 /SiO2 microspheres at 37 C
◦
and
47
°C
(b).
(a) and 47 C (b).
The diffusion rate of BSA molecules adsorbed on the surface of p-Fe3O4/SiO2 microspheres and
The diffusion rate of BSA molecules adsorbed on the surface of p-Fe O /SiO2 microspheres
the polymer networks was the fastest, faster than for molucules absorbed3 in4 the mesoporous
or
and the polymer networks was the fastest, faster than for molucules absorbed in the mesoporous or
polymer network of the microspheres. Thus, below the VPTT, the loaded BSA molecules on the
polymer network of the microspheres. Thus, below the VPTT, the loaded BSA molecules on the surface
surface of p-Fe3O4/SiO2 microspheres and polymer networks were released into the solution via
of p-Fe3 O4 /SiO2 microspheres and polymer networks were released into the solution via diffusion
diffusion (Supplementary Information Figure S6). However, above the VPTT, the BSA was released
through two routes: diffusion and shrinkage extrusion. The BSA absorbed in the mesoporous or
polymer network of the microspheres can be “squeezed out” when the temperature-sensitive polymer
Materials 2017, 10, 411
12 of 14
(Supplementary Information Figure S6). However, above the VPTT, the BSA was released through two
routes: diffusion and shrinkage extrusion. The BSA absorbed in the mesoporous or polymer network
of the microspheres can be “squeezed out” when the temperature-sensitive polymer transforms from
a swollen hydrophilic state into a contracted hydrophobic state. Therefore, the cumulative release rate
was higher at 47 ◦ C.
4. Conclusions
Thermoresponsive P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres with surface holes have been
prepared by self-assembly of bowl-like hollow p-Fe3 O4 /SiO2 microspheres with P(NIPAM-AA).
In this paper, p-Fe3 O4 /SiO2 microspheres had a cavity size of ~60 nm, surface holes of 18.3~37.2 nm,
BET surface area about 39.6 m2 /g and porosity up to 76%. Mesopores (4–12 nm) were also formed
in the p-Fe3 O4 /SiO2 microspheres after oxalic acid etching for 30 min and 45 min. The VPTT of the
prepared P(NIPAM-AA)/Fe3 O4 /SiO2 microspheres was 42.5 ◦ C, which can meet the requirements
for hyperthermia treatment, and the Ms was 72.7 emu/g which is very high in the current material.
The BSA loading capacity can be improved after P(NIPAM-AA) was grafted inside of the cavity and
outside of the p-Fe3 O4 /SiO2 microspheres. The BSA loading and release behaviors of microspheres
were studied, and they suggest that the microspheres might have potential biomedical applications.
Supplementary Materials: The following are available online at www.mdpi.com/1996-1944/10/4/411/s1.
Acknowledgments: This work was supported by grants from NSFC (project No. 21103052), the Specialized
Research Fund for the Doctoral Program of Higher Education in China (20114433110001) and the Specialized
Research Fund for the Doctoral Program of Higher Education of China (20114433110001).
Author Contributions: Xiaoyi Fu and Minqiang Xie designed the study. Jing Zhao, Ming Zeng and
Kaiqiang Zheng performed the experiments. Jing Zhao wrote this article. Xinhua He reviewed and edited
the manuscript. All authors read and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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