Materials Research Bulletin 45 (2010) 654–658

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Materials Research Bulletin

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Short communication

Bismuth subcarbonate nanoparticles fabricated by water-in-oil microemulsion-

assisted hydrothermal process exhibit anti-Helicobacter pylori properties
Rong Chen a,b,*, Gang Cheng a, Man Ho So b, Jiliang Wu a, Zhong Lu a, Chi-Ming Che b, Hongzhe Sun b,**
a
Key Laboratory for Green Chemical Process of Ministry of Education and School of Chemical Engineering & Pharmacy, Wuhan Institute of Technology, Xiongchu Street, Wuhan,
PR China
b
Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong,
Pokfulam Road, Hong Kong, PR China

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

Article history: Well-crystallized bismuth subcarbonate ((BiO)2CO3) nanoparticles were successfully synthesized by
Received 4 July 2009 water-in-oil (w/o) microemulsion-assisted hydrothermal method. In this synthesis, bismuth citrate and
Received in revised form 9 December 2009 urea are used as the precursors and the thermal decomposition of citrate leads to the formation of major
Accepted 24 December 2009
carbonate anion. The reaction, nucleation and growth steps are confined inside the water droplets and
Available online 11 January 2010
hence uniform and well-crystallized spherical nanoparticles are formed. Importantly, these
nanoparticles exert comparable anti-Helicobacter pylori activities to the clinically used drug, colloidal
Keywords:
bismuth subcitrate (CBS), which offers a promise for development of new nanomedicines.
A. Inorganic compounds
A. Nanostructures
ß 2010 Elsevier Ltd. All rights reserved.
C. Electron microscopy
C. X-ray diffraction

1. Introduction been reported to exhibit antibacterial activities [12,13]. In our

Bismuth compounds such as colloidal bismuth subcitrate (CBS)
and ranitidine bismuth citrate (RBC) have widely been used in
clinic for the treatment of gastrointestinal disorders and Helico-
bacter pylori (H. pylori) infection together with antibiotics [1,2].
Bismuth subcarbonate (BSC) has also been shown to be effective
against this bacterium [3].
Recently, there has been an increasing interest in biological
applications of various nanoparticles. Near infrared (NIR) sensitive
nanoparticles such as Au–Au(2)S nanoparticles are intensively being
developed for biomedical applications including drug and gene
delivery [4,5]. The uses of nanoparticles as antibacterial agents such
as silver based nanoparticles which are found to be effective against
E. coli, S. aureus and Ps. aeruginosa have been reported [6–9]. The
proteomic analysis of the mode of antibacterial action of silver
nanoparticles was also investigated [10,11]. Metal oxide based
nanoparticles, e.g. magnesium oxide and titanium dioxide have also
* Corresponding author at: Key Laboratory for Green Chemical Process of
Ministry of Education and School of Chemical Engineering & Pharmacy, Wuhan
Institute of Technology, Xiongchu Street, Wuhan, PR China. Tel.: +86 13 659815698;
fax: +86 27 87194465.
** Corresponding author at: Department of Chemistry and Open Laboratory of
Chemical Biology of the Institute of Molecular Technology for Drug Discovery and
Synthesis, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China.
Tel.: +852 28598974; fax: +852 28571586.
E-mail addresses: rchenhku@hotmail.com (R. Chen), hsun@hku.hk (H. Sun).
previous study, bismuth subcarbonate nanotubes have also been
reported to exhibit antibacterial properties against H. pylori [14].
Conventionally, water-in-oil (w/o) microemulsion method
has been widely employed in the synthesis of nanoparticles
owing to the spherical size-confined reverse micelles [15]. These
reverse micelles have almost the same size, leading to the
formation of uniform, mono-dispersed nanoparticles [16].
Various nanoparticles such as TiO2, Bi, CdS, Pt, Pd and Rh, have
been synthesized using the w/o microemulsion method [17–24].
SnO2 nanoparticles synthesized by hydrothermal process with
the aid of w/o microemulsion were also reported [25]. Recently,
nano-scale yttrium–barium–copper oxide (Y2BaCuO5, Y211)
particles were obtained by using water-in-oil (w/o) microemul-
sion method and nanoparticles of AgBr have been prepared by
precipitating AgBr in the water pools of microemulsions
consisting of CTAB, n-butanol, isooctane and water [26,27]. To
further increase antimicrobial activity of bismuth subcarbonate
and possibly explore new bismuth drugs, bismuth subcarbonate
(BiO)2CO3 nanoparticles were synthesized by water-in-oil (w/o)
microemulsion-assisted hydrothermal process using bismuth
citrate as a precursor.
In the present work, the w/o microemulsion was prepared by a
CTAB/n-pentanol/n-hexane/water system with the ratio of
CTAB:n-pentanol:water to be 1:2:7. CTAB was used as a surfactant
while n-pentanol as a cosurfactant. It was found that the (BiO)2CO3
nanoparticles exhibit antibacterial properties against H. pylori (50%
inhibition at 10 mg/mL), a bacterium causing peptic ulcers and

0025-5408/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.materresbull.2009.12.035
 R. Chen et al. / Materials Research Bulletin 45 (2010) 654–658
gastritis. To our knowledge, this is the first report of bismuth
subcarbonbate nanoparticles.
2. Experimental
2.1. Materials
Bismuth citrate and urea were purchased from Aldrich.
Cetyltrimethylammonium bromide (CTAB) was purchased from
Lancaster. n-Pentanol and n-hexane were purchased from Ajax
Finechem. All the reagents were analytical grade and used directly
without further purification.
2.2. Synthesis
In a typical experiment, 3.64 g CTAB (10 mmol, 0.1 M) was
dissolved in 9.0 mL n-pentanol (83 mmol) and 89.7 mL n-hexane
(0.69 mol) in a 100 mL round bottom flask, followed by addition of
1.3 mL of 50 mM bismuth citrate (65 mmol) to the oil phase. The
mixture was stirred for 15 min to form a yellow transparent
microemulsion. 0.66 g urea (10 mmol) was added to the mixture
with vigorous stirring for 12 h in order to dissolve all the urea. The
mixture was then sonicated for 30 min, and transferred to a Teflon-
lined stainless steel autoclave to perform hydrothermal process at
150 8C for 4 h. After the hydrothermal process, the solid products
were collected and washed with ethanol and water several times
by centrifugation. The samples were finally dried in a desiccator for
few days at room temperature for further characterization.
2.3. Characterization
The structures of the obtained products were characterized by
X-ray powder diffraction (XRD) using Philips PW1830 powder X-
ray diffractometer with graphite monochromatized Cu Ka radia-
tion (l = 1.540562 Å) and nickel filter. The scanning rate is
0.58 min1 in the 2u range from 20 to 708. X-ray photoelectron
spectroscopy (XPS) of the solid product was performed on a
Physical Electronic 5PHI 5600 with monochromatic Al Ka X-ray
source. The microstructures and morphologies were analyzed with
selected area electron diffraction (SAED), transmission electron
microscopy (TEM) and high-resolution transmission electron
microscopy (HRTEM), which were carried out on a Philips Tecnai
20 electron microscope using an accelerating voltage of 200 kV.
Energy-dispersive X-ray microanalysis was applied to investigate
the component, which was performed on an Oxford Instruments
INCA with a scanning range from 0 to 20 keV.
2.4. Biological studies and measurement
H. pylori 26695 (a gift from Li Ka Shing Faculty of Medicine, the
University of Hong Kong) was cultured on Brucella agar (Difco)
plates supplemented with 10% defibrinated sheep blood at 37 8C
under microaerophilic conditions (5% CO2, 4% O2 and 91% N2),
maintained by Campypak Plus (BBL). A series of bismuth
compounds with different concentrations were added to each
well which contains the same amount of H. pylori suspensions and
cultured at 37 8C under microaerophilic conditions (5% CO2, 4% O2
and 91% N2) for 3–4 days. Together with the control, the optical
density at 600 nm (OD600) of each well was measured and the
bacterial survival percentages were determined.
3. Result and discussion
The as-synthesized bismuth products were first characterized
by powder XRD analysis and the result was depicted in Fig. 1a. The
powder XRD pattern showed strong diffraction peaks, which could
655
be matched to the standard data of tetragonal (BiO)2CO3 (JCPDS No.
41-1488). Broadening of diffraction peaks indicated the small
crystal size exhibited by (BiO)2CO3 nanoparticles since the line-
width is inversely proportional to the size of nano-crystallite [28].
The presence of few weak unidentified diffraction peaks were
attributed to the thimbleful impurities. X-ray photoelectron
spectroscopy (XPS) was used to evaluate the oxidation state and
composition of the solid bismuth sample (Fig. 1b). In the high-
resolution X-ray photoelectron spectra (XPS), two strong peaks in
the Bi region at 158.7 and 164.0 eV were assigned to the Bi(4f)
binding energy (Fig. 1c). The peaks in the carbon region at 284.6
and 287.9 eV and oxygen region at 530.0 eV correspond to the
C(1s) and O(1s) transition respectively (Fig. 1d and e). The ratio of
the peak integrations was found to be near 2:1:5 for Bi, C and O,
respectively.
Fig. 2 shows the TEM (a and b) and high-resolution TEM (c and
d) images of (BiO)2CO3 nanoparticles prepared by w/o micro-
emulsion-assisted with hydrothermal process. Spherical and
nearly uniform nanoparticles can be observed in the whole
sample, Fig. 2a and b. The range of the particle size varies from 5 to
15 nm, with an average diameter and standard deviation to be
about 9.2 and 1.0 nm (see Fig. S1). The particle size distribution of
these nanoparticles is relatively narrow, indicative of uniform
(BiO)2CO3 nanoparticles being formed. It is in good agreement with
the estimated nanoparticle size from the XRD data (12.6 nm).
Lattice fringes can be clearly observed from the HRTEM images of
(BiO)2CO3 nanoparticles, suggesting that the (BiO)2CO3 nanopar-
ticles are well-crystallized and have a high order of crystallinity
(Fig. 2c and d). The interplanar distance (d-spacing) is about
3.723 Å, in well agreement with the literature value of (0 1 1) plane
of tetragonal (BiO)2CO3 (3.72 Å, JCPDS No. 41-1488). The corre-
sponding SAED pattern of selected individual (BiO)2CO3 nanopar-
ticles showed orderly arranged spots, which is a typical diffraction
pattern for single crystal structure materials, again indicative of a
high order of crystallinity (Fig. 2c, inset). The inset of Fig. 2b shows
the corresponding SAED pattern which is performed at a selected
area of (BiO)2CO3 nanoparticles. It exhibits several diffraction ring
patterns, which is characteristic for a polycrystallized structure.
This is consistent with the result of a single crystal structure of
individual (BiO)2CO3 nanoparticles deduced previously. The
observed SAED pattern in Fig. 2b, is the sum of different single-
crystallized (BiO)2CO3 nanoparticles since each of them show a
different orientation and crystal plane. Energy-dispersive spec-
troscopy (EDX) was also used to analyze the elemental composi-
tions of these (BiO)2CO3 nanoparticles (see Fig. S2). The presence of
bismuth, carbon and oxygen peaks is obviously attributed to the
(BiO)2CO3 nanoparticles, while the presence of carbon coupled
with copper peaks is probably corresponding to the carbon coated
copper grid.
It has been reported previously that bismuth nitrate pentahy-
drate and urea were the precursors in the synthesis of bismuth
oxide nanoparticles [29]. The reaction involves thermal hydrolysis
of urea to produce hydroxide (OH) [25,30] and subsequent
condensation of Bi(OH)3 intermediate to give rise to the desired
Bi2O3 (Eqs. (1)–(3)):
D
ðNH2 Þ2 CO þ 3H2 O!CO2 þ 2NHþ
4 þ 2OH

(1)
Bi3þ þ 3OH ! BiðOHÞ3 (2)
D
2BiðOHÞ3 !Bi2 O3 þ 3H2 O (3)
Interestingly, (BiO)2CO3 nanoparticles were obtained when
bismuth citrate, instead of bismuth nitrate, was used as the
substrate in this study. The only difference is the incorporation of
citrate anions in the substrate comparing with the previous
656 R. Chen et al. / Materials Research Bulletin 45 (2010) 654–658

Fig. 1. Powder XRD spectrum of (BiO)2CO3 nanoparticles (a) and XPS spectra of (BiO)2CO3 nanoparticles at low resolution and high resolution (b) at bismuth (Bi(4f), c), carbon
(C(1s), d) and oxygen (O(1s), e) region.

studies, therefore, a possible source of the carbonate anions in the
product may come from the thermal decomposition of citrate
anions. Whereas the carbonate anions generated may then replace
one of the oxide anions in Bi2O3 and contribute to the formation of
(BiO)2CO3. Therefore, the bismuth citrate acts not only as bismuth
precursor but also as the source of carbonate and oxygen.
The water-in-oil (w/o) microemulsion-assisted hydrothermal
method may play an important role for fabrication of well-
separated (BiO)2CO3 nanoparticles. The water-in-oil (w/o) micro-
emulsion provides a size-confined nanoreactor for nucleation and
growth. It is a thermodynamically stable heterogeneous system in
which water droplets with sizes less than 100 nm, are dispersed in
the oil phase [31,32]. The surfactant molecules surrounding the
water droplets, facilitate the formation of spherical reverse
micelles with uniform size at nano-scale [33], leading to
formation of a uniform shape and size of (BiO)2CO3 nanoparticles,
as illustrated in Fig. 3. In the microemulsion system, the anionic
bismuth citrate and polar urea dissolves in the polar water
droplets. Before hydrothermal process, continuous coalescence
and decoalescence of water droplets due to Brownian motion lead
to the even distribution of the substrates among the water
droplets [16]. This ensures the high local reactant concentration
and results in fast nucleation, which further leads to rapid
formation of small and substantial amount of nuclei [25]. During
 R. Chen et al. / Materials Research Bulletin 45 (2010) 654–658 657

Fig. 2. TEM (a and b) and HRTEM (c and d) images of (BiO)2CO3 nanoparticles. The insets of (b) and (c) show the corresponding SAED pattern of the selected area of
nanoparticles and individual nanoparticle, respectively.

the hydrothermal process, a high pressure and high temperature
environment is established inside the Teflon vessel. Under this
condition, a supercritical phase or near-supercritical phase may
be achieved. Compounds can therefore be well crystallized at this
region and give the expected single-crystallized nanoparticles
[34]. The synthesis of nanomaterials using supercritical fluid has
been well documented [35,36]. Once the colloidal particles are
formed, the surfactant molecules will immediately cap on the
surface of the particles and prevent them from coagulation. Upon
removal of the solvent, these capped nanoparticles will coagulate
together due to the Van der Waal interaction between the
hydrocarbon tails. This may explain why some (BiO)2CO3
nanoparticles are surrounded by some amorphous materials in
the TEM images.

Fig. 4. Inhibition profiles of (BiO)2CO3 nanoparticles (*) together with bulk

Fig. 3. Schematic diagram showing the proposed growth mechanism of (BiO)2CO3 (BiO)2CO3 (*), colloidal bismuth subcitrate (&) and Bi2O3 nanoparticles (~)
nanoparticles by w/o microemulsion-assisted with hydrothermal process. toward H. pylori.
658 R. Chen et al. / Materials Research Bulletin 45 (2010) 654–658

To evaluate the antimicrobial activity of these nanoparticles, References

their inhibitory activities against H. pylori were evaluated by a
[1] G.G. Briand, N. Burford, Chem. Rev. 99 (1999) 2601.
standard method. The minimum inhibitory concentration (MIC) was [2] J.R. Lambert, P. Midolo, Aliment. Pharmacol. Ther. 11 (1997) 27.
determined by measuring the OD values after incubation for 3 days. [3] K. Vogt, M. Warrelmann, H. Hahn, Zentralbl. Bakteriol. 273 (1990) 33.
The inhibition was found to be bismuth concentration dependent for [4] G.M. Chow, M.C. Tan, L. Ren, J.Y. R. Ying. US Patent, Appl. Publ. (2006) 32.
[5] X.L. Huang, B. Zhang, L. Ren, S.F. Ye, L.P. Sun, Q.Q. Zhang, M.C. Tan, G.M. Chow,
(BiO)2CO3 nanoparticles: >85% inhibition at 80 mg/mL of (BiO)2CO3 Mater. Med. 19 (2008) 2581.
nanoparticles; 65% at 20 mg/mL and 50% at 15 mg/mL (Fig. 4). The [6] I. Sondi, B.S. Sondi, J. Colloid Interface Sci. 275 (2004) 177.
IC50 value was evaluated to be 10 mg/mL. The anti-H. pylori activities [7] S. Zhang, R. Fu, D. Wu, W. Xu, Q. Ye, Z. Chen, Carbon 42 (2004) 3209.
[8] L. Balogh, D.R. Swanson, D.A. Tomalia, G.L. Hagnauer, A.T. McManus, Nano. Lett. 1
of colloidal bismuth subcitrate (CBS), bulky (BiO)2CO3 and bismuth (2001) 18.
oxide nanoparticles were also examined under identical conditions [9] G.A. Martı́nez-Castañón, N. Niño-Martı́nez, F. Martı́nez-Gutierrez, J.R. Martı́nez-
for comparison. The inhibitory activity of CBS was about half of the Mendoza, F. Ruiz, J. Nanopartic. Res. 10 (2008) 1343.
[10] C.N. Lok, C.M. Ho, R. Chen, Q.Y. He, W.Y. Yu, H. Sun, P.K.-H. Tam, J.F. Chui, C.-M.
(BiO)2CO3 nanoparticles. Moreover, the anti-H. pylori activity of bulk
Che, J. Proteome. Res. 5 (2006) 916.
(BiO)2CO3 was about 1/3 of the nanoparticles. It indicated that [11] C.N. Lok, C.M. Ho, R. Chen, Q.Y. He, W.Y. Yu, H. Sun, P.K.-H. Tam, J.F. Chui, C.-M. Che,
(BiO)2CO3 nanoparticles exhibit slightly enhanced and comparable J. Bio. Inorg. Chem. 12 (2007) 527.
inhibitory properties compared with the bulky (BiO)2CO3 and the [12] P.K. Stoimenov, R.L. Klinger, G.L. Marchin, K.J. Klabunde, Langmuir 18 (2002)
6679.
clinically used antiulcer drug, colloidal bismuth subcitrate (CBS), [13] S.Y. Kwak, S.H. Kim, Environ. Sci. Technol. 35 (2001) 2388.
respectively. In contrast, almost no inhibitory properties were found [14] R. Chen, M.H. So, J. Yang, F. Deng, C.-M. Che, H. Sun, Chem. Commun. (2006)
for bismuth oxide nanoparticles. 2265.
[15] B.L. Cushing, V.L. Kolesnichenko, C.J. O’Connor, Chem. Rev. 104 (2004) 3893.
[16] K. Holmberg, J. Colloid Interface Sci. 274 (2004) 355.
4. Conclusion [17] Y.Y. Mori, Y. Okastu, Y. Tsujimoto, J. Nanopartic. Res. 3 (2001) 219.
[18] J. Fang, K.L. Stokes, J.A. Wiemann, W.L. Zhou, J. Dai, F. Chen, C.J. O’Connor, Mater.
Sci. Eng. B 83 (2001) 254.
In conclusion, well-crystallized (BiO)2CO3 nanoparticles with [19] A. Agostiano, M. Catalano, M.L. Curri, M. Della Monica, L. Manna, L. Vasanelli,
an average particle size of 9.2 nm were synthesized via the w/o Micron 31 (2000) 253.
microemulsion-assisted hydrothermal method using bismuth [20] H. Härelind Ingelsten, J.-C. Béziat, K. Bergkvist, A.E.C. Palmqvist, M. Skoglundh,
Q.H. Hu, L.K.L. Falk, K. Holmberg, Langmuir 18 (2002) 1811.
citrate as the precursor. Importantly, these nanoparticles exhibited
[21] K. Persson, P.O. Thevenin, K. Jansson, J. Agrell, S.G. Järås, L.J. Pettersson, Appl. Catal.
a comparable anti-H. pylori activities to the clinically used drug, A 249 (2003) 165.
colloidal bismuth subcitrate. These studies are likely to provide a [22] M. Kishida, T. Hanaoka, W.Y. Kim, H. Nagata, K. Wakabayashi, Appl. Surf. Sci. 121–
122 (1997) 347.
basis for further development of multiple treatments of H. pylori
[23] T. Hanaoka, T. Hatsuta, T. Tago, M. Kishida, K. Wakabayashi, Appl. Catal. A 190
infection or new nanomedicines. (2000) 291.
[24] M. Kishida, K.-i. Ichiki, T. Hanaoka, H. Nagata, K. Wakabayashi, Catal. Today 45
Acknowledgements (1998) 203.
[25] D. Chen, L. Gao, J. Colloid Interface Sci. 279 (2004) 137.
[26] F. Li, C. Vipulanandan, J. Nanopartic. Res. 9 (2007) 841.
This work was supported by the National Natural Science [27] M.M. Husein, E. Rodil, J.H. Vera, J. Nanopartic. Res. 9 (2007) 787.
Foundation of China (Grant 20801043), Wuhan Chenguang [28] E.N. Kaufmann, Characterization of Materials, vol. 2, John Wiley & Sons, Wiley-
Interscience, 2003.
Scheme (Grant 200850731376) established under Wuhan Science [29] R.K. Jha, R. Pasricha, V. Ravi, Ceram. Int. 31 (2005) 495.
and Technology Bureau and the Area of Excellence Scheme (AoE/P- [30] R.J.M.J. Vogels, J.T. Kloprogge, J.W. Geus, J. Colloid Interface Sci. 285 (2005)
10/01) established under the University Grants Committee of the 86.
[31] L. Garcia-Rio, P. Hervella, Chem. Eur. J. 12 (2006) 8284.
Hong Kong Special Administrative Region China. [32] O. Sonneville-Aubrun, D. Babayan, D. Bordeaux, P. Lindner, G. Rata, B. Cabane,
Phys. Chem. Chem. Phys. 11 (2009) 101.
[33] R.J. Stokes, D.F. Evans, Fundamentals of Interfacial Engineering, 1st ed., Wiley-
Appendix A. Supplementary data VCH, New York, United States of America, 1997, 240.
[34] Y. Xia, P. Yang, Y. Sun, Y. Wu, B. Mayers, B. Gates, Y. Yin, F. Kim, H. Yan, Adv. Mater.
15 (2003) 353.
Supplementary data associated with this article can be found, in [35] J. Otsu, Y. Oshima, J. Supercrit. Fluids 33 (2005) 61.
the online version, at doi:10.1016/j.materresbull.2009.12.035. [36] J.A. Darr, M. Poliakoff, Chem. Rev. 99 (1999) 495.