Journal of

Materials Chemistry B
PAPER

Hexagonal boron nitride nanoplates as emerging

biological nanovectors and their potential
Cite this: J. Mater. Chem. B, 2016,
4, 6103 applications in biomedicine†
Tun Lu,a Libo Wang,a Ye Jiang,a Qiuwen liub and Caijin Huang*b

Received 16th June 2016,
Accepted 16th August 2016
DOI: 10.1039/c6tb01481j
www.rsc.org/MaterialsB
The application of nanomaterials in the biological and medical areas has attracted great attention.
Cytotoxicity, stability and solubility are the prerequisites for a nanomaterial to be considered for application in the
field of biomedicine. Here, we suggest a simple method to produce highly dispersed water-soluble ultrathin
h-BN nanoplates whose size measures ca. 30–60 nm in diameter and 1.6 nm in thickness. Moreover, we
demonstrate that h-BN nanoplates can act as a reliable biological nanovector to carry proteins by cross-linking
immobilization. Furthermore, the biocompatibility of h-BN nanoplates has also been explored via an apoptosis
assay. In addition, a successful attempt has been made to investigate the potency of h-BN nanoplates as an
immunostimulating adjuvant in a mouse immunization experiment. Preliminary results show that the level of
antibody response stimulated by an antigen protein (bovine serum albumin) linked with h-BN is ca. 4 times
higher than that by the antigen protein alone. This work gives evidence that water-soluble h-BN nanoplates are
of high biocompatibility and low reactogenicity and therefore they can serve as an excellent biomedical
platform for nanoparticle–biomolecular interactions. They preserve and even enhance the bioacitivities of
the cross-linked antigen proteins, which strongly suggests their use in nanoparticle vaccine design.

1. Introduction
The rapid development of nanotechnology makes it possible to
engineer materials precisely at the nanoscale level, which meets
the growing demands of biological and medical fields.1–5 In
particular, the application of nanoparticles to biology and medicine
improves traditional diagnostic, imaging and therapeutic
techniques,6–10 and provides a powerful tool for the exploration
and understanding of physiological processes as well.11–14 This
multifunctionality of nanoparticles benefits from their unique
physicochemical properties: first, they have a larger specific
surface area than the bulk for biomacromolecules (e.g. peptide,
protein and DNA) to be bound on. Second, a controllable
design in the composition, size, shape and surface properties
of nanoparticles affords more opportunities in biological and
medical applications. Third, nanoparticles are generally much
smaller in dimension than host cells, and can enter living
cells by cellular endocytosis or cell membrane penetration.
Many types of nanoparticles, such as liposomes, carbon materials,
a
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350002,
China
b
State Key Laboratory of Photocatalysis on Energy and Environment,
College of Chemistry, Fuzhou University, Fuzhou 350002, China.
E-mail: cjhuang@fzu.edu.cn
† Electronic supplementary information (ESI) available: Size distribution, zeta
potential and protein-release assay data. See DOI: 10.1039/c6tb01481j
gold, silver and calcium phosphate, have been well introduced
into bio-related fields and play an important role in diagnosis,
bio-imaging and therapy by carrying biomolecules, biomarkers
and drugs in living systems.6,15,16 For example, carbon nano-
tubes have been found to be good vectors by surface functiona-
lization for the transport of biomolecules.17,18 Veiseh et al.
demonstrated that magnetic iron nanoparticle carriers can track
siRNA delivery and improve tumor specificity and potency for
cancer therapy.19 Gold nanoparticles supported chitosans have
been successfully used as vehicles for the delivery of plasmid
DNA with a serum antibody response 10 times more powerful
than the naked DNA vaccine.20 However, to be a good biomedical
nanovector, aqueous solubility and stability are required for
interacting with or transporting molecules of interest. Besides,
cytotoxicity and biocompatibility are crucial factors for nano-
materials to be considered in biosystems. Therefore, high solubility,
biocompatibility and low cytotoxicity are urgently desired while
hunting for a suitable biomedical nanomaterial.
Recently, hexagonal boron nitride (h-BN), a structural analogue
of graphite in nature, has attracted great attention due to its unique
physical and chemical properties.21 As a two-dimensional layered
material, it is reasonable to use h-BN as support materials,
e.g. functional nanomaterials22,23 with improved performance.
Moreover, since h-BN has high chemical stability and very low
toxicity, it seems favorable for h-BN to be exploited as a
biomaterial in living systems. Additionally, the colorlessness

This journal is © The Royal Society of Chemistry 2016 J. Mater. Chem. B, 2016, 4, 6103--6110 | 6103
Paper Journal of Materials Chemistry B

of h-BN is convenient for the detection of optical signals from

proteins, DNA and other molecules of interest. However, the
extremely poor solubility of boron nitride materials (including
boron nitride nanotubes) is a major obstacle that limits their
integration into biological systems. To overcome this limitation,
coating materials (e.g. polymers24) can be used to improve their poor
solubility to some extent, but this has the risks of: (1) decoration of
the coating polymer in vivo and thus causing unexpected effects and
(2) high concentration of the coating polymer inducing intrinsic
cytotoxicity. Therefore, for h-BN nanomaterials, both solubility and
biosafety remain challenging key factors for their realistic biomedical
applications. We propose herein a new type of bio-vehicle—h-BN
nanoplates—synthesized by one-step thermal decomposition of
urea and boric acid in the presence of copper nitrate. Our work
demonstrated that h-BN nanoplates could be highly dispersed in
water to form high-quality colloids without any coating poly-
mers. Furthermore, h-BN nanoplates were covalently loaded with Fig. 1 Structure characterization of h-BN nanoplates. (a) XRD patterns,
proteins and further explored in the mouse immune system as inset: photo of the h-BN nanoplate colloid. (b) FT-IR spectrum. (c) Raman
biological nanovectors and adjuvants. Adjuvants are materials spectrum and (d) SEM image of the as-prepared h-BN nanoplates.
which can strongly promote antibody response to a defined
antigen in a nonspecific manner,25 which are especially useful
matrix and subsequently being taken up by dendritic cells.
for weakly immunogenic new-generation vaccines (e.g. synthetic
Moreover, milky and colloidal h-BN nanoplates (Fig. 1, inset) at
peptides, recombinant proteins and DNA). Bovine serum albumin
concentrations up to 20 mg mL1 were successfully prepared
(BSA) was used as a model immunogenic protein (antigen) to
without any co-solvents, ensuring their transportation and
evaluate the adjuvanticity of h-BN nanoplates. The h-BN
circulation in living systems. The absence of co-solvents, especially
nanoplate-supported BSA (BSA@BN) stimulated a high immune
macromolecules, avoided the risk of their decoration in vivo or in
response in mouse experiments and showed high biocompatibility
the cells and their intrinsic bio-toxicity. Meanwhile, Fig. S2 (ESI†)
and low reactogenicity, which indicated great potential for the use
shows the zeta potential of the h-BN nanoplate colloid
in biomedical engineering such as biomolecular transporters and
(0.02 mg mL1). The zeta potential of h-BN nanoplates in
in vaccine design.
neutral water was ca. 30 mV, suggesting high stability of the
as-prepared h-BN colloid.

2. Results and discussion 2.2 Cross-linking of h-BN nanoplates and BSA

2.1 Preparation and characterization of h-BN nanoplates
h-BN nanoplates were prepared through pyrolysis of urea and
B2O3 in the presence of Cu(NO3)2 under an NH3 atmosphere.
Fig. 1a shows the X-ray diffraction pattern of the obtained
sample. We can see a good agreement between the diffraction
peaks and the standard data (JCPDF Card No. 34-0421) for
hexagonal boron nitride. The FT-IR spectrum of the as-obtained
sample (Fig. 1b) presents the two peaks located at 1386 cm1
and 813 cm1, which are considered to be the fingerprints of
h-BN.22 The small peak at 3400 cm1 originates from the
groups –OH and –NH2, which is important for the anchoring
of the desired antigen/protein on the surface of adjuvants.26
Fig. 1c demonstrates the typical Raman spectrum of h-BN with
a sharp peak at 1368 cm1 representing B–N stretching vibra-
tion attributed to the E2g phonon mode.22 The XPS survey
spectra (Fig. 2) display the peak at 190.5 eV in the B1s spectrum
and the peak at 398.2 eV in the N1s spectrum, indicating the
formation of B–N bonds.27 The as-prepared h-BN exhibits plate-
like morphology (Fig. 1d and 3a) with a size ca. 30–60 nm in
diameter (Fig. S1, ESI†) and ca. 1.6 nm in thickness (Fig. 3c and d).
As reported,28 nanoparticles with the sizes of 10–100 nm are
considered to be favorable for penetrating the extracellular
Bovine serum albumin (BSA) was used not only as a model
protein for interaction with h-BN nanoplates but also as an
antigen for the subsequent immune response. The typical
procedure of the cross-linking of BSA and h-BN nanoplates is
illustrated in Fig. 4a. The surface of h-BN nanoplates was first
pre-treated with ammonium hydroxide and then functionalized
with glutaraldehyde (GA), a typical protein cross-linking
reagent.29 In the chemical cross-linking reaction, GA was used
as an amino group to form a Schiff base, predominantly with
the z-amino group of lysine.30,31 BSA was then grafted through
GA on the surface of h-BN nanoplates to form a layer of antigen.
To verify the cross-linking of BSA and h-BN nanoplates, UV-Vis
spectra, fluorescence spectra and element mapping were ana-
lyzed. As shown in Fig. 4b, a characteristic peak located at
280 nm was found for BSA alone32 in comparison with a
monotonic variation for h-BN. However, while BSA was con-
jugated with h-BN nanoplates, a blue shift took place for the
characteristic peak of BSA due to the modification of BSA by
conjugation.33 The conjugation between BSA and h-BN could
also be confirmed by the emission spectra of the fluorescein
isothiocyanate (FITC)-labeled BSA (FITC-BSA). FITC served
as a chromophore marker for determining/analyzing the

6104 | J. Mater. Chem. B, 2016, 4, 6103--6110 This journal is © The Royal Society of Chemistry 2016
Journal of Materials Chemistry B Paper

Fig. 3 Surface morphology of h-BN nanoplates. (a) Low- and (b) high-
magnification TEM images, inset: high-resolution TEM image. (c) AFM
image of h-BN nanoplates. (d) Height profile of the white line marked in (c).

Fig. 2 XPS survey spectra of the as-obtained h-BN powder, specifying

B and N 1s core levels, respectively. The spectrum curves (blue) are
deconvoluted by Gaussian fitting. Elemental formula and possible bonding
information are given in the spectra.

FITC-associated proteins. FITC-BSA was used to replace BSA in

the cross-linking reaction to prepare BSA-FITC coated h-BN
nanoplates (FITC-BSA@BN). The characteristic emission peak
centered at 520 nm for FITC34 could be found for the sample
FITC-BSA@BN (Fig. 4c), indicating the binding of BSA to the
surface of h-BN nanoplates. Furthermore, the anchoring of BSA
on h-BN nanoplates could be observed by the electron micro-
graphs. Element mapping showed that boron atoms (contained
Fig. 4 Cross-linking of BSA and h-BN nanoplates. (a) Schematic descrip-
in h-BN) and sulfur atoms (contained in BSA) were widely
tion of the procedure of cross-linking. (b) UV-Vis spectra of BSA, h-BN and
distributed on the surface of the sample (Fig. 4d–f), illustrating BSA@BN. (c) Emission spectra of h-BN and BSA@BN excited at 490 nm.
that sulfur-containing BSA was highly dispersed and conjugated (d) SEM image of the as-prepared BSA@BN. (e and f) Boron and sulfur
with h-BN nanoplates. From this, we can conceive that h-BN element mapping of the area boxed in (d), respectively.
nanoparticles may conjugate with other biomolecules (e.g.
DNA and RNA) with an appropriate linker as reported for
other inorganic nanoparticles.35,36 Furthermore, Fig. S3 (ESI†) 2.3 The protein loading capacity of h-BN nanoplates
presents the in vitro release of BSA from h-BN nanoparticles. The Loading capacity is an important capability for bio-vectors to
results showed only o6% of the protein BSA was released from carry biomolecules such as proteins, DNA and RNA. BSA is used
nanoparticles at pH 7.4, confirming the high stability of the as the model protein to evaluate the protein loading capacity of
cross-linking of BSA and nanoparticles. h-BN nanoplates. In the experiment, instead of BSA, FITC-BSA

This journal is © The Royal Society of Chemistry 2016 J. Mater. Chem. B, 2016, 4, 6103--6110 | 6105
Paper
was used owing to the quantifiable fluorescence properties of
FITC. The amount of h-BN-linked FITC-BSA can be calculated
by subtracting the amount of unreacted FITC-BSA from the
total amount of FITC-BSA added into the system. Therefore, the
BSA-loading capacity (LC) of the nanoplates can be calculated
by the following equation:
Total amount BSA  unassociated amount BSA
LC ¼  100
Nanoparticles weight
(1)
Fig. 5 shows the emission spectra of a series of given FITC-BSA
solutions for the standard curve and the unassociated FITC-BSA
solution. The cross-linked FITC-BSA measured 0.19 mg per
1 mg h-BN, which was a roughly estimated amount for the
cross-linked BSA. As a result, the BSA-loading capacity for h-BN
nanoplates was 19%. From this value, we can also speculate that
each h-BN nanoplate (diameter: 60 nm; height: 1.6 nm; density:21
2.34 g cm3) linked about 18 BSA molecules. Furthermore, for a
Fig. 5 (a) UV-Vis spectra of a series of FITC-BSA solutions at different
concentrations and the centrifugal supernatant after the cross-linking
reaction. (b) Standard curve for measuring the concentration of the
unassociated FITC-BSA in the supernatant. Data originated from peaks
intensity at 520 nm in (a). The concentration of the unassociated FITC-BSA
(dashed line) in the supernatant was 0.037 mg mL1.
6106 | J. Mater. Chem. B, 2016, 4, 6103--6110
Journal of Materials Chemistry B
nanoparticle biovector, the particle size/volume is crucial for its
transport in living systems, so we propose a definition of BSA-
loading density (LD) for an individual nanoplate as follows:
Associated BSA amount of individual nanoparticles
LD ¼
Nanoparticle volume
(2)
Taking 18 BSA molecules bound to each h-BN nanoplate (diameter:
60 nm; height: 1.6 nm), we can calculate that the BSA-loading
density of an individual nanoplate is 4  103 nm3 (4  106 mm3).
2.4 Evaluation of immune response
The antibody known as immunoglobulin (Ig), is produced by
plasma cells and used by the immune system to identify and
neutralize pathogens such as bacteria and viruses. The antibody
response in the serum depends on the living system’s immune
response to the antigen. The stronger antibody response
indicates stronger immunocompetence of the antigen—a hall-
mark of a good vaccine candidate. Fig. 6a shows that two weeks
after the BSA@BN injection, the antibody response increased
significantly compared to those injected with BSA alone (with
a two-sided t-test result of P(t) o 0.02); for the secondary
immunization, BSA@BN once again induced significantly
higher antibody response compared to those using BSA alone
Fig. 6 Immune response of BSA@BN. (a) Antibody concentrations in
the mice injected with h-BN, BSA and BSA@BN, respectively. P(t) o 0.02.
(b) IFN-g concentrations in the mice injected with h-BN, BSA and BSA@BN.
P(t) o 0.001.
This journal is © The Royal Society of Chemistry 2016
Journal of Materials Chemistry B Paper

(with a two-sided t-test result of P(t) o 0.02). The average level

of anti-BSA antibody in the mouse serum immunized with
BSA@BN was ca. 4 times higher than those immunized with
BSA alone. This clearly demonstrates that the linked h-BN
nanoplates greatly enhanced the immunocompetence of the
coupled antigen, just as an adjuvant does in a vaccine formula.
Cytokine interferon g (IFN-g) is of importance in determining the
effectiveness of the immune response to antigens/pathogens.
IFN-g belongs to a large class of proteins known as cytokines
which are responsible for the communication between cells and
for triggering the defensive immune system that helps to eradicate
pathogens.37 The production of IFN-g by KM mice is shown in
Fig. 6b. Before immunization, the levels of IFN-g were very low and
below the detection limit. After the stimulation of BSA or BSA@BN,
the production of IFN-g was dramatically enhanced, especially for
those immunized with BSA@BN (P(t) o 0.001).
Levels of both antibody and IFN-g in mice after BSA@BN
treatment were higher than BSA alone, which demonstrates
that the immunocompetence of the antigen protein BSA was
significantly enhanced by auxiliary compound h-BN nanoplates.

2.5 The biocompatibility of h-BN nanoplates

Biocompatibility is pivotal for biomaterials to exert their high
performance and reduce unacceptable degree of damage to the
body health. Although low/non-toxicity has been mentioned in
the literature concerning boron nitride materials, especially
boron nitride nanotubes,24,38–40 few reports pay attention to the
bio-safety of h-BN nanosheets. In this work, we extended our
investigation on bio-safety of h-BN nanoplates to gain more
insights. Fig. 6 shows that no apparent antibody response was
Fig. 8 Biocompatibility of h-BN nanoplates. Influence of (a) the control
and (b) h-BN nanoplates on the apoptotic rate of CHO cells detected by
flow cytometry.
observed by the injection of h-BN nanoplates alone in this study,
implying that it is non-immunogenic in vivo. Moreover, during
the course of the trial, no apparent physiological reactions
(e.g. swelling, ecthyma) were observed at the site of injection,
implying that h-BN nanoplates were well tolerated in mice and
exhibited low reactogenicity. Furthermore, the results from
apoptosis assay demonstrate that the influence of h-BN nano-
plates on the apoptotic rate of HEK-293T cells (Fig. 7) or CHO
cells (Fig. 8) was slight by comparing the early apoptotic cells in
the sample and in the control. Moreover, as depicted in Fig. 9,
after 48 h of incubation with HEK-293T and CHO cells, the MTT
values were significantly steady at the concentrations of h-BN in
the assessed range (0–100 mg mL1). Thus, h-BN nanoplates can
be considered biocompatible at appropriate concentrations,
which encourages their further applications in living systems.

Fig. 7 Biocompatibility of h-BN nanoplates. Influence of (a) control and
(b) h-BN nanoplates on the apoptotic rate of HEK-293T cells detected by
flow cytometry.
3. Conclusions
In summary, a simple method of synthesizing h-BN nanoplates
was reported using urea and boric acid in the presence of
copper nitrate at a high temperature. The obtained h-BN
nanoplates measured ca. 30–60 nm in diameter and 1.6 nm
in thickness. Moreover, our experiment showed that an aqueous
colloid of h-BN nanoplates could be successfully prepared with-
out stabilizer/co-solvent, indicating their potential applications
to nanoparticle–biomolecular interaction. Furthermore, the
results demonstrated that water-soluble h-BN nanoplates could
act as a good nanovector to load a model protein BSA by covalent
conjugation with GA. This approach provides a reliable way for

This journal is © The Royal Society of Chemistry 2016 J. Mater. Chem. B, 2016, 4, 6103--6110 | 6107
Paper
Fig. 9 MTT assays on (a) HEK-293T and (b) CHO cell cultures incubated
for 48 h with different concentrations of h-BN nanoplates. Results are
presented as mean value  standard error; n = 3 for the MTT assays.
h-BN nanoplates to carry/transport biomolecules such as proteins,
DNA and RNA in a similar fashion for living systems or biosensing
platforms. Finally, we found that h-BN-supported BSA can
stimulate a higher level of immune response than BSA alone
in mice, showing that h-BN nanoplates maintained and even
enhanced the bioactivity-immunocompetence of the cross-
linked protein. This suggests that the h-BN nanoplates play the
role of adjuvants for the antigen protein bound and can be used
in vaccine design. To further explore their usage in biomedical
applications, the biocompatibility of h-BN nanoplates was
evaluated by apoptosis assay and non-cytotoxicity was found.
In addition, their low reactogenicity was observed by no adverse
reactions induced at the site of injections. All these findings
show that h-BN nanoplates hold great promise for their applica-
tions as biological nanovectors, biocompatible materials and
vaccine adjuvants.
4. Methods
4.1 Preparation of h-BN nanoplates
Typically, boron oxide, urea and copper nitrate were mixed at a
mass ratio of 100 : 600 : 9. The mixture was heated to 1250 1C
for 5 hours under an ammonia atmosphere. The as-obtained
red powder was ground and then washed with nitric acid by
6108 | J. Mater. Chem. B, 2016, 4, 6103--6110
Journal of Materials Chemistry B
vigorously stirring until the formation of a white precipitate.
The white powder was then washed with deionized water until a
milky colloid was formed.
4.2 Preparation of the antigen (BSA)-conjugated h-BN
nanoplates
The antigen-coated h-BN nanoplates were prepared via a cross-
linking reaction as described below: in short, h-BN nanoplates
were pretreated by ammonium hydroxide, and the excess of
ammonium hydroxide was removed by dialysis. Equal volumes
(10 mL) of BSA (1 mg mL1) and h-BN nanoplate colloid
(0.02 mg mL1) were mixed well, followed by adding glutar-
aldehyde at a final concentration of 0.1% (v/v). The mixture was
kept at room temperature for 2 h.41 The cross-linking reaction was
stopped by dialysis in MilliQ water to remove glutaraldehyde.
4.3 Characterization of the samples
Powder X-ray diffraction (XRD) patterns of the samples were
obtained using a Bruker D8 Advance diffractometer using
Cu Ka 1 radiation at room temperature. X-ray photoelectron
spectra (XPS) were collected on a SHIMADZU (Amicus), using
Mg Ka X-ray as the excitation source (1486.8 eV). Fourier
transform infrared spectroscopy (FTIR) was performed on a
nicolet 670 FT-IR spectrometer where KBr was used as the
transmission standard and the diluting reagent. Raman spectra
were recorded from 500 to 2500 cm1 on a Renishaw inVia
Raman Microphrobe at 633 nm laser excitation. UV/Vis spectra
were read on a Varian Cary 500 Scan UV/Vis system. The
morphologies and microstructures of the samples were
explored using field emission scanning electron microscopy
(SEM, S-450, Hitachi, Japan), a transmission electron micro-
scopy (TEM) (TECNAI F30) operated at 200 kV and an atomic
force microscopy (AFM) (Agilent SPM5500). The zeta potential
was measured using a laser particle size and zeta potential
analyzer (BI-200SM, BROOKHAVEN).
4.4 Cross-linking of FITC-labeled BSA and h-BN nanoplates
To evaluate the amount of cross-linked BSA, 0.8 mg mL1
fluorescein isothiocyanate (FITC)-labeled BSA (FITC-BSA) (Sangon
Biotech Co., Ltd, Shanghai, China) was used instead of BSA in the
cross-linking reaction to prepare BSA-FITC coated h-BN nanoplates
(FITC-BSA@BN). The as-obtained FITC-BSA@BN colloid was kept
in the dark for 8–12 h after the cross-link reaction was initiated with
FITC-BSA and h-BN colloid (0.08 mg mL1) (v/v = 1 : 4), and then the
colloid was centrifuged at 10 000 g for 5 min and re-suspended with
MilliQ water. The supernatant was used for the quantification of
unassociated FITC-BSA. To verify that the cross-linking took place
between BSA and h-BN nanoplates, fluorescence spectra were
recorded on a Perkin Elmer LS55 luminescence spectrometer. To
evaluate the amount of the cross-linked BSA, the emission spectra
of FITC-BSA@BN were measured using a fluorescence spectro-
photometer (Varian Cary Eclipse) with an excitation at 490 nm.
4.5 Mice and vaccination regimen
KM male mice (aged 4 weeks) were purchased from Shanghai
SLAC laboratory Animal Co., Ltd. The mice were housed at the
This journal is © The Royal Society of Chemistry 2016
Journal of Materials Chemistry B
laboratory animal center of the Fujian University of Traditional
Chinese Medicine. They were fed a standard laboratory pellet
diet and pure water ad libitum, and the animal housing room
was maintained in the range of 24 to 26 1C with 55 to 75%
humidity under 12 h light/dark cycles. The animals were
acclimatized for one week before they were used in experiments.
All animal experiments were approved by the Fujian Institute of
Experimental Animals Committee (Publication SYXK2009-0001).
All of the mice were randomly divided into 4 groups of 10 ones
and vaccinated twice at 0 and 2 weeks. BSA (100 mg) or BSA@BN
(containing 100 mg BSA) was conducted via intraperitoneal
injection. The control group was injected with MilliQ water.
The animals were boosted after 14 days with the same formula-
tion. Unless otherwise stated, 100 mg BSA was administered
following intraperitoneal injection. Serum samples were taken
before inoculation at 14, 28 days post-inoculation.
4.6 Antibody responses
The anti-BSA antibodies were detected by ELISA according to
the procedures proposed by Fan.42 Briefly, 96-well microtiter-
plates were coated with 100 mL BSA (dissolved in carbonate
buffer (50 mM, pH 9.6), 10 mg mL1) overnight at 4 1C. The wells
were washed three times with phosphate buffer saline (PBS)
containing 0.05% Tween20 (PBST) and blocked with 200 mL
PBST containing 1% (w/v) gelatin, followed by incubation at
37 1C for 1 h. After three washes with PBST, the as-obtained
serum (100 mL) was serially diluted in PBST containing 0.1% (w/v)
gelatin and incubated at 37 1C for 1 h. The wells were washed
three times with PBST and then added with the second anti-
body polyclonal goat anti-mouse IgG conjugated horse radish
peroxidase (HRP). After four washes with PBST, 100 mL tetra-
methylbenzidine (TMB) was added to each well. After incuba-
tion in the dark at room temperature for 20 min, 50 mL H2SO4
(0.5 M) was added to each well to stop chromophore production.
The quantification of the mouse anti-BSA antibodies can be
monitored by measuring the absorbance read at 450 nm, which
was carried out on a microplate reader for ELISA (Shanghai Kehua
Bio-engineering Co., Ltd). When the value ratio of the sample
serum to the control serum was higher than 2.1, the corres-
ponding anti-BSA activity was considered to be positive.
4.7 Cytokine ELISA assays
The presence of IFN-g in mouse serum at the 4th week after the
first immunization was measured using IFN-g Enzyme Linked
Immunosorbent Assay (ELISA) kits (Boshide, Wuhan, China).
The amount of IFN-g was estimated by comparison against a
standard curve of recombinant rat IFN-g.
4.8 Cells and cell cultures for apoptosis assay
Human embryonic kidney cells (HEK-293T) and Chinese hamster
ovary cells (CHO) were maintained at 37 1C under a humidified
atmosphere with 5% CO2, in the DMEM cell culture medium
supplemented with 10% fetal calf serum and 1% double-
resistance penicillin–streptomycin (all from Beijing TransGen
This journal is © The Royal Society of Chemistry 2016
Paper
Biotech Co., Ltd). The cells were prepared for experiments
using the conventional trypsinization procedure with trypsin.
4.8.1 Apoptosis assay detected by flow cytometry. The cells
were incubated in flat-bottom 6-well (1  105 cells per well) cell
culture plates. h-BN nanoplates were added into the wells at the
final concentration of 20 mg mL1. Sterilized double distilled
water was used instead of h-BN nanoplates for the control. After
being rested for 48 h, cell cultures were treated with trypsin
without EDTA, and then cells were collected and washed with
PBS. After being added with 1 annexin V binding buffer,
annexin V-FITC and PI, the cells were read using flow cytometry
(BD FACS Calibur).
4.8.2 MTT assays. The cells were seeded in 96-well plate
chambers (5000 cells per well). Once adhesion was verified
(about 12 h after seeding), cells were incubated with 0, 20, 40,
60, 80, and 100 mg mL1 of h-BN for 48 h, and at the endpoint,
with MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium
bromide, C0009 from Beyotime) 0.5 mg mL1 for further 4 h.
After cell treatment with 100 mL of DMSO (dimethyl sulfoxide,
C0009 from Beyotime) for another 4 h, the absorbance at 562 nm
was measured using a microplate reader (ELx800, BioTek).
Appropriate controls were performed incubating cells with the
sterilized double distilled water.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (no. 2014BAC13B03, 21273038, 21543002
and 11305091) and the Natural Science Foundation of Fujian
Province, China (no. 2014J01225).
References
1 V. Saini, V. P. Zharov, C. S. Brazel, D. E. Nikles, D. T. Johnson
and M. Everts, Nanomed. Nanotechnol. Biol. Med., 2006, 2,
200–206.
2 J. Kerssemakers, L. Ionov, U. Queitsch, S. Luna, H. Hess and
S. Diez, Small, 2009, 5, 1732–1737.
3 T. Hideyo, T. Hideyuki, M. R. Kevin, B. K. Madhukar,
K. N. Siva, K. Kazuhiro, F. Parviz and R. B. Eric, Nanotech-
nology, 2011, 22, 245101.
4 A. Chałupniak, E. Morales-Narváez and A. Merkoçi, Adv.
Drug Delivery Rev., 2015, 95, 104–116.
5 L. P. Lee, IEEE Int. Electron Devices Meet., 2010, 1.3.1–1.3.5.
6 N. Sanvicens and M. P. Marco, Trends Biotechnol., 2008, 26,
425–433.
7 W. Lin, Chem. Rev., 2015, 115, 10407–10409.
8 L. Zhang, F. Gu, J. Chan, A. Wang, R. Langer and
O. Farokhzad, Clin. Pharmacol. Ther., 2008, 83, 761–769.
9 Y. Lu, Q. Hu, Y. Lin, D. B. Pacardo, C. Wang, W. Sun,
F. S. Ligler, M. D. Dickey and Z. Gu, Nat. Commun., 2015,
6, 10066.
10 H. Wang, P. Agarwal, S. Zhao, J. Yu, X. Lu and X. He, Nat.
Commun., 2015, 6, 10081.
J. Mater. Chem. B, 2016, 4, 6103--6110 | 6109
Paper
11 S. Pradhan, P. Patra, S. Mitra, K. K. Dey, S. Basu, S. Chandra,
P. Palit and A. Goswami, J. Agric. Food Chem., 2015, 63,
2606–2617.
12 J. Yasur and P. Usha Rani, Chemosphere, 2015, 124, 92–102.
13 C.-Y. Wu, D. Young, J. Martel and J. D. Young, Nanomedicine,
2015, 10, 2659–2676.
14 S. A. Stanley, J. Sauer, R. S. Kane, J. S. Dordick and
J. M. Friedman, Nat. Med., 2015, 21, 92–98.
15 T. Mohan, P. Verma and D. N. Rao, Indian J. Med. Res., 2013,
138, 779–795.
16 Y. Liu, Y. Xu, Y. Tian, C. Chen, C. Wang and X. Jiang, Small,
2014, 10, 4505–4520.
17 P. Wu, X. Chen, N. Hu, U. C. Tam, O. Blixt, A. Zettl and
C. R. Bertozzi, Angew. Chem., Int. Ed., 2008, 120, 5100–5103.
18 A. Bianco, K. Kostarelos, C. D. Partidos and M. Prato, Chem.
Commun., 2005, 571–577.
19 O. Veiseh, F. M. Kievit, C. Fang, N. Mu, S. Jana, M. C. Leung,
H. Mok, R. G. Ellenbogen, J. O. Park and M. Zhang, Bio-
materials, 2010, 31, 8032–8042.
20 X. Zhou, X. Zhang, X. Yu, X. Zha, Q. Fu, B. Liu, X. Wang,
Y. Chen, Y. Chen and Y. Shan, Biomaterials, 2008, 29,
111–117.
21 R. Haubner, M. Wilhelm, R. Weissenbacher and B. Lux, in
High Performance Non-Oxide Ceramics II, ed. M. Jansen,
Springer, Berlin, Heidelberg, 2002, ch. 1, vol. 102, pp. 1–45.
22 C. Huang, W. Ye, Q. Liu and X. Qiu, ACS Appl. Mater.
Interfaces, 2014, 6, 14469–14476.
23 Y. Lin, C. E. Bunker, K. S. Fernando and J. W. Connell, ACS
Appl. Mater. Interfaces, 2012, 4, 1110–1117.
24 X. Chen, P. Wu, M. Rousseas, D. Okawa, Z. Gartner, A. Zettl
and C. R. Bertozzi, J. Am. Chem. Soc., 2009, 131, 890–891.
25 M. S. Sinyakov, M. Dror, T. Lublin-Tennenbaum, S. Salzberg,
S. Margel and R. R. Avtalion, Vaccine, 2006, 24, 6534–6541.
6110 | J. Mater. Chem. B, 2016, 4, 6103--6110
Journal of Materials Chemistry B
26 Q. Weng, B. Wang, X. Wang, N. Hanagata, X. Li, D. Liu,
X. Wang, X. Jiang, Y. Bando and D. Golberg, ACS Nano, 2014,
8, 6123–6130.
27 C. Huang, C. Chen, X. Ye, W. Ye, J. Hu, C. Xu and X. Qiu,
J. Mater. Chem. A, 2013, 1, 12192–12197.
28 L. Zhao, A. Seth, N. Wibowo, C.-X. Zhao, N. Mitter, C. Yu and
A. P. Middelberg, Vaccine, 2014, 32, 327–337.
29 F. M. Richards and J. R. Knowles, J. Mol. Biol., 1968, 37,
231–233.
30 K. Peters and F. M. Richards, Annu. Rev. Biochem., 1977, 46,
523–551.
31 A. Habeeb and R. Hiramoto, Arch. Biochem. Biophys., 1968,
126, 16–26.
32 B. Chakraborty and S. Basu, J. Lumin., 2009, 129, 34–39.
33 D. Hopwood, C. Allen and M. McCabe, Histochem. J., 1970,
2, 137–150.
34 K. Aslan, M. J. R. Previte, Y. Zhang and C. D. Geddes, J. Phys.
Chem. C, 2008, 112, 18368–18375.
35 Z. Liu, S. M. Tabakman, Z. Chen and H. Dai, Nat. Protoc.,
2009, 4, 1372–1381.
36 C. Kneuer, M. Sameti, E. G. Haltner, T. Schiestel, H. Schirra,
H. Schmidt and C.-M. Lehr, Int. J. Pharm., 2000, 196,
257–261.
37 J. Parkin and B. Cohen, Lancet, 2001, 357, 1777–1789.
38 A. Aydoğdu and N. Sevinç, J. Eur. Ceram. Soc., 2003, 23,
3153–3161.
39 W. Lei, D. Portehault, R. Dimova and M. Antonietti, J. Am.
Chem. Soc., 2011, 133, 7121–7127.
40 G. Ciofani, S. Danti, G. G. Genchi, B. Mazzolai and
V. Mattoli, Small, 2013, 9, 1672–1685.
41 F. Richards and J. Knowles, J. Mol. Biol., 1968, 37, 231–233.
42 H. Fan, S. Xiao, T. Tong, S. Wang, L. Xie, Y. Jiang, H. Chen
and L. Fang, Mol. Immunol., 2008, 45, 653–660.
This journal is © The Royal Society of Chemistry 2016