- Email: [email protected]
Extracellular biosynthesis of copper sulfide nanoparticles by Shewanella oneidensis MR-1 as a photothermal agent
Accepted Manuscript Title: Extracellular biosynthesis of copper sulfide nanoparticles by Shewanella oneidensis MR-1 as a photothermal agent Author: Nan-Qing Zhou Li-Jiao Tian Yu-Cai Wang Dao-Bo Li Pan-Pan Li Xing Zhang Han-Qing Yu PII: DOI: Reference:
S0141-0229(16)30055-2 http://dx.doi.org/doi:10.1016/j.enzmictec.2016.04.002 EMT 8893
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
6-3-2016 31-3-2016 1-4-2016
Please cite this article as: Zhou Nan-Qing, Tian Li-Jiao, Wang Yu-Cai, Li Dao-Bo, Li Pan-Pan, Zhang Xing, Yu Han-Qing.Extracellular biosynthesis of copper sulfide nanoparticles by Shewanella oneidensis MR-1 as a photothermal agent.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2016.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript
Extracellular biosynthesis of copper sulfide nanoparticles by Shewanella oneidensis MR-1 as a photothermal agent
Nan-Qing Zhoua, Li-Jiao Tiana, Yu-Cai Wangb, Dao-Bo Lia, Pan-Pan Lib , Xing Zhanga, Han-Qing Yua a
Department of Chemistry, bSchool of Life Sciences, University of Science and Technology of China, Hefei, 230026, China
*Corresponding author: Prof. Han-Qing Yu, Fax: +86 551 63601592; E-mail: [email protected]
1
1
Abstract
2
Photothermal therapy (PTT) is a minimally invasive and effective cancer
3
treatment method and has a great potential for innovating the conventional
4
chemotherapy approaches. Copper sulfide (CuS) exhibits photostability, low cost, and
5
high absorption in near infrared region, and is recognized as an ideal candidate for
6
PTT. However, CuS, as a photothermal agent, is usually synthesized with traditional
7
chemical approaches, which require high temperature, additional stabilization and
8
hydrophilic modification. Herein, we report, for the first time, the preparation of CuS
9
nanoparticles as a photothermal agent by a dissimilatory metal reducing bacterium
10
Shewanella. oneidensis MR-1. The prepared nanoparticles are homogenously shaped,
11
hydrophilic, small-sized (~5 nm) and highly stable. Furthermore, the biosynthesized
12
CuS nanoparticles display a high photothermal conversion efficiency of 27.2%
13
because of their strong absorption at 1100 nm. The CuS nanoparticles could be
14
effectively used as a PTT agent under the irradiation of 1064 nm. This work provide a
15
simple, eco-friendly and cost-effective approach for fabricating PTT agents.
16 17
Keywords: Shewanella oneidensis; biosynthesis; copper sulfide; photothermal
18
therapy
2
19
Introduction
20 21
Photothermal therapy (PTT) refers to the employment of appropriate agents for
22
effectively ‘burning’ the cancer cells without damaging the healthy tissue. Such a
23
treatment approach has attracted growing interests in recent years, and an increasing
24
number of compounds have been explored as PTT candidates. Four types of agents
25
have been reported to exhibit photothermal effects [1], including noble metal
26
nanostructures (Au [2], Pd [3]), carbon-based nanomaterials (carbon nanotubes [4],
27
graphene [5]), organic nanoparticles (NPs) like polymeric NPs [6], polypyrole NPs
28
[7]), and semiconductor nanostructures (W18O49 [8], CuS [9], Cu2-xSe [10], MoS2
29
[11] ). Among them, the noble metal nanostructures are most widely used because of
30
their relatively high photothermal conversion efficiency and low toxicity.
31
Nevertheless, the high cost of noble metal limits their broad application. In addition,
32
the main optical absorption contributed by localized plasmon resonance in noble
33
metals locates on the visible region, which brings about no benefit for in vivo
34
treatment. Compared to visible light, near-infrared (NIR) laser is much less absorbed
35
by biological tissues, and thus can penetrate several centimeters through the
36
epidermis [11]. Therefore, PTT agents with strong adsorption in the NIR (λ =
37
700-1100 nm) region and low costs are highly desired.
38
CuS, as a cheap semiconductor with strong absorption in NIR region, has been
39
extensively used for catalysis [12], chemical sensors and superionic materials in the
40
past. Recently, CuS NPs have attracted increasing interests from biomedical 3
41
researchers due to their intriguing properties. Thus, their application in PTT has been
42
explored [13,14]. Also, efforts have been devoted to fabricate CuS NPs with distinct
43
morphologies, e.g., flower like [9] and plate like [15], using various precursors and
44
methodologies. In the most of these approaches, such as hot injection, hydrothermal
45
synthesis and thermal decomposition, high temperature is required, and toxic
46
hydrogen sulfide (H2S) is released during the synthesis process. Furthermore, the
47
chemically generated nanomaterials are usually hydrophobic and exhibit a high
48
toxicity to cells when applied in biomedicine. As an alternate synthesis route, the
49
biosynthesis process relies on the reducing power of microorganisms, rather than
50
toxic organic reagents, to assemble metal ions into stable nanocrystallites. Thus,
51
microbial synthesis of NPs is environmentally friendly and cost-effective. So far,
52
numerous microorganisms including yeast, fungi and bacteria have been used as a
53
green nanofactory for producing different types of NPs, e.g., Au [16], Pd [17], CdSe
54
[18], PbS [19], etc. Although there have been a few reports on biofabricating CuS by
55
fungus [20,21], the biosynthesis of CuS NPs by bacteria has not been reported yet.
56
Shewanella oneidensis is a widely distributed Gram-negative γ-proteobacterium,
57
featured with diverse periplasmic and membrane-inserted reductases. This bacterium
58
possesses various electron pathways and secrets mediator like flavins that strengthen
59
the bioreduction of extracellular electron acceptors. It can use various terminal
60
electron acceptors including oxygen (O2), nitrate (NO3-), thiosulfate (S2O32-), ferric
61
iron (Fe3+), manganese (Mn4+) and uranium (U6+) [22]. S. oneidensis has been widely
62
studied for the bioremediation of environmental contaminants and bioenergy 4
63
generation. It is also used for the biosynthesis of sulfur-containing semiconductor NPs
64
such as As2S3 [23], ZnS [24], Ag2S [25] through reducing thiosulfate to sulfide.
65
In this work, we developed an environmentally benign method to extracellularly
66
synthesize small-sized, high stability, and well-dispersed CuS NPs by using S.
67
oneidensis MR-1 as a nanofactory under ambient temperature and pressure. Then, the
68
biofabricated CuS NPs were isolated and characterized in terms of the structure,
69
crystal phase, chemical composition and photo property. Furthermore, the
70
photothermal performance of bio-CuS NPs was also evaluated.
71 72
Materials and Methods
73 74
Biosynthesis of CuS NPs
75
S. oneidensis MR-1 was cultured in Luria-Bertani broth at 30 oC with a rotary
76
shaker (200 rpm) for 16 h until the late stationary phase. The cells were harvested by
77
centrifugation (6000×g, 4 oC, 7 min) and washed two times with sterile sodium
78
4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffered mineral
79
medium [26]. The aseptic HEPES-buffered mineral medium that was prepared under
80
anaerobic conditions by purging with pure N2 was used as the growth medium.
81
Sodium lactate (20 mM) served as the carbon source and electron donor, while
82
Na2S2O3 (1mM) was applied as the electron acceptor. The initial optical density (OD)
83
at 600 nm was about 0.1. After 24 h of growth (30 oC, 200 rpm), CuCl2 (1 mM) was
84
injected into the medium. Followed by incubation for another 24 h, the mixture was 5
85
centrifuged (8000×g, 10 min) to remove the cells, and the supernatant was
86
concentrated by ultrafiltration (10,000 Da) to collect the CuS NPs. The concentrated
87
supernatant was washed thrice by distilled water and then freeze dried for the
88
characterizations of X-ray powder diffraction (XRD), X-ray photoelectron
89
spectroscopy (XPS), and thermal stability.
90 91
Characterization
92
The morphology of the biosynthesized CuS NPs were imaged by high-resolution
93
transmission electron microscopy (HRTEM) (JEM-2000, JEOL Co., Japan) with an
94
electron kinetic energy of 200 kV. The structure of NPs was obtained from X-ray
95
powder diffraction (XRD) (TTR-III, Rigaku Co., Japan). The thermal stability of the
96
bio-CuS NPs was characterized with a DTG-60H/DSC-60 thermogravimetric analyzer
97
(Shimadzu Co., Japan) under N2 atmosphere. The heating range was from 10 oC to
98
800 oC at a rate of 10 oC per min. The chemical composition and the valence states of
99
component elements were analyzed by X-ray photoelectron spectroscopy (XPS)
100
(ESCALAB250, Thermo Fisher Inc., USA). UV-vis adsorption spectra analysis was
101
performed
102
(Shimadzu Co., Japan).
on
a
UV-3600
ultraviolet-visible-near-infrared
spectrophotometer
103 104 105 106
Photothermal property measurement To study the photothermal performance of the bio-CuS NPs, a series concentration of samples were irradiated under a 1064 nm laser (Inter-Diff Co., China) 6
107
with an output of 0.32 W for 8 min, and naturally cooled to room temperature
108
afterwards. The infrared camera was used to record the temperature variation of
109
different samples. The concentration of Cu2+ was measured by inductively coupled
110
plasma atomic emission spectroscopy (ICP-AES, PerkinElmer Inc., USA).
111 112
In vitro photothemal therapy evaluation
113
Nanoparticles tend to aggregate in vivo, thus they will be cleaned by the immune
114
system. For this reason, when nanoparticles are used for therapy or drug delivery in
115
biomedical field, surface modification is usually necessary to strengthen its
116
dispersibility. Generally polyethylene glycol (PEG) is commonly used as a modifier
117
because of its good biocompatibility and high efficiency to obstruct the aggregation of
118
nanoparticles. In order to fully simulate the practical case in the cancer therapy, a PEG
119
modification was adopted when evaluating the photothemal therapy of CuS NPs. The
120
obtained bio-CuS NPs were embellished with polyethylene glycol methelether
121
pre-modified by zinc sulfate before test. The concentration of modifier was 2 times to
122
that of the CuS NPs. After the dose of modifier, the mixture was maintained in 37 oC
123
for 24 h.
124
The in vitro photothermal therapy of the bio-CuS NPs was evaluated using the
125
methyl thiazolyl tetrazolium (MTT) assay in human pulmonary carcinoma cell lines
126
(A549R) [27]. A549R cells were seeded into a 96-well plate (5 × 103 cells per well)
127
and cultured at 37 oC and 5% CO2 for 24 h before use. Then, the culture medium was
128
removed and replaced by medium containing CuS NPs at different concentrations, 7
129
followed by further 24-h incubation. After the incubation, the cells were exposed to
130
the 1064 nm laser for 10 min (1.5 W/cm2). Subsequently, 100 µL of MTT (1 mg/mL)
131
was added to each well of the 96-well plate and reacted at 37 oC and 5% CO2 for 4 h.
132
After the reaction, 15% sodium dodecyl sulfate of 150 µL was added into the wells.
133
The assay plate was maintained overnight at room temperature, and the microplate
134
reader was applied to measure the absorbance of each well at 492 nm.
135 136 137
Results and Discussion
138 139
Biosynthesis and characteristics of the bio-CuS NPs
140
S. oneidensis accumulates S2- in the reducing medium with S2O32- as the electron
141
acceptor. After the addition of 1 mM CuCl2 and 24-h anaerobic incubation, the color
142
of the medium became brown. After centrifugation, the supernatant remained brown,
143
indicating that the CuS NPs were biosynthesized extracellularly and could be easily
144
isolated (Fig. S1). The brown-colored supernatant was collected by centrifugation and
145
characterized using TEM. The TEM image (Fig. 1a) shows that the obtained CuS NPs
146
had a uniform particle size of ~5 nm. The color of the resulting NPs was not
147
consistent with that of CuS NPs reported before (black or dark green) [28,29] , which
148
might be attributed to their tiny size. The CuS NPs showed a high solubility in water
149
with a solute concentration up to 600 µg/mL. The size of the water-dispersed CuS
150
NPs was around 14 nm, as determined by Zeta Sizer (Fig. S2). The larger size of 8
151
nanoparticles in water dispersion is attributed to the aggregation of the
152
high-concenrtation CuS NPs in solution. Microstructure information of the bio-CuS
153
nanoparticles was obtained from HRTEM. The HRTEM image (Fig. 1b) displays the
154
lattice fringe with a d-spacing of 0.304 nm, which is in agreement with the (022)
155
interplanar spacing of CuS.
156
In order to identify the phase structure of the NPs, we concentrated the
157
supernatant by ultrafiltration under anaerobic conditions. The collected samples were
158
freeze-dried for XRD analysis. The XRD pattern (Fig. 1c) of the as-synthesized
159
nanoparticles involved several characteristic peaks, such as (022), (023) and (130),
160
which were further determined to be the standard CuS phase (JCPDS card no.
161
65-7111). The XRD pattern result corresponds with the HRTEM images well.
162
Chemical composition of the bio-CuS NPs
163
Since the chemical composition of bio-synthesized NPs critically affects their
164
physical performance, thermogravimetric analysis and XPS spectra measurement
165
were conducted. The thermal degradation behavior of the bio-induced CuS NPs is
166
shown in Fig. 2. The weight loss of the sample started at 45 oC, and only 1.7% of the
167
total weight was lost below 100 oC, which could be referred to free water.
168
Subsequently, there was a major degradation peak from 98 oC to 465 oC in the
169
thermogravimetry analysis (TGA) curve, and the 16.8% weight loss suggests the
170
transformation of CuS to Cu2S. The valence states of Cu and S in the samples were
171
determined by XPS spectra. The peaks at 932.1 eV and 951.9 eV indicate Cu 2p3/2 and
172
Cu 2p1/2, respectively (Fig. 3a). In Fig. 3b, the sample displayed a slightly broad and 9
173
asymmetric peak at about 162.2 eV, while the S 2p3/2 peak position of 162.0 eV is
174
assigned to thiolate moiety (Cu-S bonds). The asymmetric XPS spectra can be fitted
175
into S 2p3/2 peak at 162.0 eV and S 2p1/2 peak at 163.5 eV. The other weaker peak
176
appeared at 168.4 eV, indicating the 2p3/2 chemical shift of a sulfonate (SO32-) [30].
177
This shift could be ascribed to the disproportionation of S2O32-. The quantification
178
analysis of XPS spectra shows the atomic ratio of Cu : S was 0.94 : 1 (Table S1),
179
which is in agreement with the ratio of CuS.
180 181
Photothermal effect of the bio-CuS NPs
182
To measure the optical property of the CuS NPs dispersed in water, the sample
183
(480 µg/mL) was analyzed by UV-vis-NIR spectroscopy. The spectrum exhibits a
184
broad-strong absorbance in the NIR region with the maximum absorption at 1100 nm
185
(Fig. 4). The peak position was different from that of the previously reported Cu2-xS
186
NPs [9,15], which might be attributed to the crystal phase diversity of individual NPs.
187
The strong NIR absorption of CuS was likely to originate from d-d intra-band
188
transitions of Cu2+, rather than the localized surface plasmon resonance. This qualifies
189
the prepared CuS a great potential in photothermal application under NIR excitation.
190
Such absorbance data inspire us to use a 1064-nm laser to test their photothermal
191
effects using a continuous irradiation with a power density of 0.53 W/cm2 for 8 min.
192
The temperature of the aqueous solution containing different concentrations (0-480
193
µg/mL) of the CuS NPs was recorded every 20 s. As shown in Fig. 5a, the samples
194
containing CuS NPs exhibited a distinguished photothermal conversion ability. With 10
195
the dose of CuS NPs (30-480 µg/mL), the temperature of CuS NPs solution increased
196
by 6-20 oC in 8 min (Fig. 5b). In contrast, the pure water exhibited a temperature
197
elevation by less than 3 oC only after the irradiation. This result demonstrates that the
198
bio-induced CuS NPs could efficiently convert the 1064 nm laser energy to thermal
199
energy.
200
The photothermal conversion efficiency of the samples was estimated using a
201
method reported by Roper et al. [31]. The conversion efficiency η of the bio-CuS NPs
202
can be calculated with Eq. (1):
203
hS (Tmax Tsur ) Q0 I (1 10 A )
(1)
204
where h is heat-transfer coefficient, S is the surface area, Tmax is the final temperature
205
after irradiation, Tsurr represents the ambient temperature, (Tmax-Tsurr) = 19.7 oC. Q0 is
206
the heat dissipated from light absorption of the solvent, which was measured
207
individually to be 11.7 mW. I is the laser power (0.53 W/cm2), and Aλ represents the
208
absorption at 1064 nm, which can be estimated to be 1.840 from Fig. 4. The value of
209
hS is derived from Eq. (2): mC hS
0 0 s 210
210
(2)
211 212
where τs is the sample system time constant, m0 and C0 represent the mass (0.3 g) and
213
specific heat capacity (4.2 J/g) of pure water, respectively.
214
Thus, the photothermal conversion efficiency of the bio-CuS NPs was calculated
215
to be 27.2%. This value is higher than that reported for Au nanorods (22%) and
216
Cu2-xSe nanocrystals (22%) [10]. 11
217
Furthermore, the photostability of the bio-CuS NPs was examined by conducting
218
four cycles of LASER ON/OFF. During each cycle, the solution of CuS NPs was
219
irradiated with the 1064 nm laser (0.53 W/cm2) for 8 min and then naturally cooled to
220
the room temperature. As shown in Fig. 7, the temperature dynamics was reproducible
221
during the cycles and showed no attenuation on the photothermal amplitude, indicating
222
that the bio-CuS NPs were highly photo-stable.
223 224
In vitro photothermal therapy of bio-CuS NPs
225
To evaluate the phtothermal therapy of the obtained CuS NPs, A549 cells were
226
incubated with the CuS NPs at different concentrations for 24 h, then exposed to 1064
227
nm laser for 10 min (1.5 W/cm2). The MTT assay was applied to determine the cell
228
viability. As shown in Fig. 8, high cell viability (> 90%) could be observed when the
229
concentration of CuS NPs was below 37.5 µg/mL without laser irradiation. However,
230
when the A549 cells were irradiated by the 1064 nm laser for 10 min, a significant
231
decrease in cell viability occurred. This results indicate that the bio-CuS NPs at a
232
relatively low concentration (37.5 µg/mL), were effective because of their high
233
photothermal conversion efficiency. This feature enables the bio-CuS to effectively kill
234
cancer cells without damaging the healthy issues, which is crucial to their biomedical
235
application.
236 237 238
Conclusions 12
239 240
In this work, small-sized (~5 nm), highly stable and hydrophilic CuS NPs with
241
distinguished photothermal conversion efficiency were successfully prepared by using
242
Shewanella oneidensis MR-1 in an eco-friendly and simple way. The biogenic CuS
243
NPs exhibited good photostability and photothermal performance with a conversion
244
efficiency up to 27.2%, higher than those of Au nanorods and Cu2-xSe nanocrystals. In
245
vitro photothermal therapy tests demonstrated the effectiveness of bio-CuS NPs,
246
ensuring bio-CuS NPs an excellent candidate for cancer treatment. In addition,
247
Shewanella oneidensis MR-1, a metal-reducing bacterium, was demonstrated to be
248
efficient in the synthesis of nanomaterials.
249 250
Acknowledgements
251
The work was supported by the Natural Science Foundation of China (21477120),
252
and the Program for Changjiang Scholars and Innovative Research Team and the
253
Collaborative Innovation Center of Suzhou Nano Science and Technology of the
254
Ministry of Education of China.
255 256
References
257 258
[1]
X. Liu, B. Li, F. Fu, K. Xu, R. Zou, Q. Wang, et al., Facile synthesis of
259
biocompatible cysteine-coated CuS nanoparticles with high photothermal
260
conversion efficiency for cancer therapy, Dalt. Trans. 43 (2014) 11709–11715. 13
261
[2]
L. Au, D. Zheng, F. Zhou, Z.-Y. Li, X. Li, Y. Xia, A quantitative study on the
262
photothermal effect of immuno gold nanocages targeted to breast cancer cells,
263
ACS Nano. 2 (2008) 1645–1652.
264
[3]
W. Fang, S. Tang, P. Liu, X. Fang, J. Gong, N. Zheng, Pd Nanosheet‐
265
Covered Hollow Mesoporous Silica nanoparticles as a platform for the chemo
266
‐photothermal treatment of cancer cells, Small. 8 (2012) 3816–3822.
267
[4]
H.K. Moon, S.H. Lee, H.C. Choi, In vivo near-infrared mediated tumor
268
destruction by photothermal effect of carbon nanotubes, ACS Nano. 3 (2009)
269
3707–3713.
270
[5]
K. Yang, L. Hu, X. Ma, S. Ye, L. Cheng, X. Shi, et al., Multimodal imaging
271
guided photothermal therapy using functionalized graphene nanosheets
272
anchored with magnetic nanoparticles, Adv. Mater. 24 (2012) 1868–1872.
273
[6]
H. Gong, L. Cheng, J. Xiang, H. Xu, L. Feng, X. Shi, et al., Near‐infrared
274
absorbing polymeric nanoparticles as a versatile drug carrier for cancer
275
combination therapy, Adv. Funct. Mater. 23 (2013) 6059–6067.
276
[7]
Z. Zha, X. Yue, Q. Ren, Z. Dai, Uniform polypyrrole nanoparticles with high
277
photothermal conversion efficiency for photothermal ablation of cancer cells,
278
Adv. Mater. 25 (2013) 777–782.
279
[8]
Z. Chen, Q. Wang, H. Wang, L. Zhang, G. Song, L. Song, et al., Ultrathin
280
PEGylated W18O49 nanowires as a new 980 nm‐laser‐driven photothermal
281
agent for efficient ablation of cancer cells in vivo, Adv. Mater. 25 (2013)
282
2095–2100. 14
283
[9]
Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, et al., Hydrophilic flower
284
‐like CuS superstructures as an efficient 980 nm laser‐driven photothermal
285
agent for ablation of cancer cells, Adv. Mater. 23 (2011) 3542–3547.
286
[10] C.M. Hessel, V. P. Pattani, M. Rasch, M.G. Panthani, B. Koo, J.W. Tunnell, et
287
al., Copper selenide nanocrystals for photothermal therapy, Nano Lett. 11
288
(2011) 2560–2566.
289
[11] S. Wang, K. Li, Y. Chen, H. Chen, M. Ma, J. Feng, et al., Biocompatible
290
PEGylated MoS2 nanosheets: Controllable bottom-up synthesis and highly
291
efficient photothermal regression of tumor, Biomaterials. 39 (2015) 206–217.
292
[12] F. Li, J. Wu, Q. Qin, Z. Li, X. Huang, Controllable synthesis, optical and
293
photocatalytic properties of CuS nanomaterials with hierarchical structures,
294
Powder Technol. 198 (2010) 267–274.
295
[13] M. Zhou, R. Zhang, M. Huang, W. Lu, S. Song, M.P. Melancon, et al., A multifunctional
[64Cu]
296
chelator-free
CuS
nanoparticle
platform
for
297
simultaneous micro-PET/CT imaging and photothermal ablation therapy, J. Am.
298
Chem. Soc. 132 (2010) 15351–15358.
299
[14] Z. Zha, S. Wang, S. Zhang, E. Qu, H. Ke, J. Wang, et al., Targeted delivery of
300
CuS nanoparticles through ultrasound image-guided microbubble destruction
301
for efficient photothermal therapy, Nanoscale. 5 (2013) 3216–3219.
302
[15] Q. Tian, F. Jiang, R. Zou, Q. Liu, Z. Chen, M. Zhu, et al., Hydrophilic Cu9S5
303
nanocrystals: A photothermal agent with a 25.7 % heat conversion efficiency
304
for photothermal ablation of cancer cells in vivo, ACS Nano. 5 (2011) 15
305
9761–9771.
306
[16] A.K. Suresh, D.A. Pelletier, W. Wang, M.L. Broich, J.-W. Moon, B. Gu, et al.,
307
Biofabrication of discrete spherical gold nanoparticles using the metal-reducing
308
bacterium Shewanella oneidensis, Acta Biomater. 7 (2011) 2148–2152.
309
[17] L. Jia, Q. Zhang, Q. Li, H. Song, The biosynthesis of palladium nanoparticles
310
by antioxidants in Gardenia jasminoides Ellis: long lifetime nanocatalysts for
311
p-nitrotoluene hydrogenation, Nanotechnology. 20 (2009) 385601.
312
[18] R. Cui, H. Liu, H. Xie, Z. Zhang, Y. Yang, D. Pang, et al., Living yeast cells as
313
a controllable biosynthesizer for fluorescent quantum dots, Adv. Funct. Mater.
314
19 (2009) 2359–2364.
315
[19] S. Seshadri, K. Saranya, M. Kowshik, Green synthesis of lead sulfide
316
nanoparticles by the lead resistant marine yeast, Rhodosporidium diobovatum,
317
Biotechnol. Prog. 27 (2011) 1464–1469.
318
[20] M. Schaffie, M.R. Hosseini, Biological process for synthesis of semiconductor
319
copper sulfide nanoparticle from mine wastewaters, J. Environ. Chem. Eng. 2
320
(2014) 386–391.
321
[21] M.R. Hosseini, M. Schaffie, M. Pazouki, E. Darezereshki, M. Ranjbar,
322
Biologically synthesized copper sulfide nanoparticles: Production and
323
characterization, Mater. Sci. Semicond. Process. 15 (2012) 222–225.
324
[22] T. Perez-Gonzalez, C. Jimenez-Lopez, A.L. Neal, F. Rull-Perez, A.
325
Rodriguez-Navarro, A. Fernandez-Vivas, et al., Magnetite biomineralization
326
induced by Shewanella oneidensis, Geochim. Cosmochim. Acta. 74 (2010) 16
327
967–979.
328
[23] J.-H. Lee, M.-G. Kim, B. Yoo, N. V Myung, J. Maeng, T. Lee, et al., Biogenic
329
formation of photoactive arsenic-sulfide nanotubes by Shewanella sp. strain
330
HN-41, Proc. Natl. Acad. Sci. 104 (2007) 20410–20415.
331
[24] X. Xiao, X.-B. Ma, H. Yuan, P.-C. Liu, Y.-B. Lei, H. Xu, et al., Photocatalytic
332
properties of zinc sulfide nanocrystals biofabricated by metal-reducing
333
bacterium Shewanella oneidensis MR-1, J. Hazard. Mater. 288 (2015)
334
134–139.
335
[25] A.K. Suresh, M.J. Doktycz, W. Wang, J.-W. Moon, B. Gu, H.M. Meyer, et al.,
336
Monodispersed biocompatible silver sulfide nanoparticles: Facile extracellular
337
biosynthesis using the γ-proteobacterium, Shewanella oneidensis, Acta
338
Biomater. 7 (2011) 4253–4258.
339
[26] D.-B. Li, Y.-Y. Cheng, C. Wu, W.-W. Li, N. Li, Z.-C. Yang, et al., Selenite
340
reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in
341
periplasm, Sci. Rep. 4 (2014).
342
[27] Y. Min, C. Mao, S. Chen, G. Ma, J. Wang, Y. Liu, Combating the drug
343
resistance of cisplatin using a platinum prodrug based delivery system, Angew.
344
Chemie Int. Ed. 51 (2012) 6742–6747.
345
[28] W. Feng, W. Nie, Y. Cheng, X. Zhou, L. Chen, K. Qiu, et al., In vitro and in
346
vivo toxicity studies of copper sulfide nanoplates for potential photothermal
347
applications, Nanomedicine Nanotechnology, Biol. Med. 11 (2015) 901–912.
348
[29] C. Zhang, Y.-Y. Fu, X. Zhang, C. Yu, Y. Zhao, S.-K. Sun, BSA-directed 17
349
synthesis of CuS nanoparticles as a biocompatible photothermal agent for
350
tumor ablation in vivo, Dalt. Trans. 44 (2015) 13112–13118.
351
[30] T. Nakanishi, B. Ohtani, K. Uosaki, Fabrication and characterization of
352
CdS-nanoparticle mono-and multilayers on a self-assembled monolayer of
353
alkanedithiols on gold, J. Phys. Chem. B. 102 (1998) 1571–1577.
354
[31] D.K. Roper, W. Ahn, M. Hoepfner, Microscale heat transfer transduced by
355
surface plasmon resonant gold nanoparticles, J. Phys. Chem. C. 111 (2007)
356
3636–3641.
357
18
Figure captions
Figure 1. (a) Low-magnification; (b) HRTEM image; and (c) XRD patterns of the bio-CuS NPs
Figure 2. Thermogravimetric analysis (TGA) of the bio-CuS NPs
Figure 3. XPS spectra of (a) Cu 2p and (b) S 2p regions of the bio-CuS NPs
Figure 4. UV-vis absorbance spectra of the bio-CuS NPs dispersed in water
Figure 5. (a) Temperature elevation of pure water and the solution of CuS NPs at different concentrations of CuS (30, 60, 120, 240, 480 µg/mL) under the irradiation of 1064 nm for 480 s; (b) plot of temperature change (ΔT) over a time period of 480 s versus the CuS concentration
Figure 6. (a) Infrared images of the CuS NPs solution (480 µg/mL) irradiated by NIR laser (1064 nm, 8 min), and then shut off; (b) temperature dynamics of the CuS NPs solution; and (c) time constant of heat transfer for the system was determined to be 161.5 s by applying the time data of cooling period vs the negative natural logarithm of driving force temperature (
T Tsurr ). Tmax Tsurr
Figure 7. Temperature elevation of the bio-CuS NPs over four LASER ON/OFF cycles
Figure 8. Cell viability of A549 cells after treatment by bio-CuS NPs at different concentrations and with different irradiation times
19
Figure 1. (a) Low-magnification; (b) HRTEM image; and (c) XRD patterns of the bio-CuS NPs
20
Figure 2. Thermogravimetric analysis (TGA) of the bio-CuS NPs
21
Figure 3. XPS spectra of (a) Cu 2p and (b) S 2p regions of the bio-CuS NPs
22
Figure 4. UV-vis absorbance spectra of the bio-CuS NPs dispersed in water
23
Figure 5. (a) Temperature elevation of pure water and the solution of CuS NPs at different concentrations of CuS (30, 60, 120, 240, 480 µg/mL) under the irradiation of 1064 nm for 480 s; (b) plot of temperature change (ΔT) over a time period of 480 s versus the CuS concentration
24
Figure 6. (a) Infrared images of the CuS NPs solution (480 µg/mL) irradiated by NIR laser (1064 nm, 8 min), and then shut off; (b) temperature dynamics of the CuS NPs solution; and (c) time constant of heat transfer for the system was determined to be 161.5 s by applying the time data of cooling period vs the negative natural logarithm of driving force temperature (
T Tsurr ). Tmax Tsurr
25
Figure 7. Temperature elevation of the bio-CuS NPs over four LASER ON/OFF cycles
26
Figure 8. Cell viability of A549 cells after treatment by bio-CuS NPs at different concentrations and with different irradiation times
27
S0141-0229(16)30055-2 http://dx.doi.org/doi:10.1016/j.enzmictec.2016.04.002 EMT 8893
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
6-3-2016 31-3-2016 1-4-2016
Please cite this article as: Zhou Nan-Qing, Tian Li-Jiao, Wang Yu-Cai, Li Dao-Bo, Li Pan-Pan, Zhang Xing, Yu Han-Qing.Extracellular biosynthesis of copper sulfide nanoparticles by Shewanella oneidensis MR-1 as a photothermal agent.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2016.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript
Extracellular biosynthesis of copper sulfide nanoparticles by Shewanella oneidensis MR-1 as a photothermal agent
Nan-Qing Zhoua, Li-Jiao Tiana, Yu-Cai Wangb, Dao-Bo Lia, Pan-Pan Lib , Xing Zhanga, Han-Qing Yua a
Department of Chemistry, bSchool of Life Sciences, University of Science and Technology of China, Hefei, 230026, China
*Corresponding author: Prof. Han-Qing Yu, Fax: +86 551 63601592; E-mail: [email protected]
1
1
Abstract
2
Photothermal therapy (PTT) is a minimally invasive and effective cancer
3
treatment method and has a great potential for innovating the conventional
4
chemotherapy approaches. Copper sulfide (CuS) exhibits photostability, low cost, and
5
high absorption in near infrared region, and is recognized as an ideal candidate for
6
PTT. However, CuS, as a photothermal agent, is usually synthesized with traditional
7
chemical approaches, which require high temperature, additional stabilization and
8
hydrophilic modification. Herein, we report, for the first time, the preparation of CuS
9
nanoparticles as a photothermal agent by a dissimilatory metal reducing bacterium
10
Shewanella. oneidensis MR-1. The prepared nanoparticles are homogenously shaped,
11
hydrophilic, small-sized (~5 nm) and highly stable. Furthermore, the biosynthesized
12
CuS nanoparticles display a high photothermal conversion efficiency of 27.2%
13
because of their strong absorption at 1100 nm. The CuS nanoparticles could be
14
effectively used as a PTT agent under the irradiation of 1064 nm. This work provide a
15
simple, eco-friendly and cost-effective approach for fabricating PTT agents.
16 17
Keywords: Shewanella oneidensis; biosynthesis; copper sulfide; photothermal
18
therapy
2
19
Introduction
20 21
Photothermal therapy (PTT) refers to the employment of appropriate agents for
22
effectively ‘burning’ the cancer cells without damaging the healthy tissue. Such a
23
treatment approach has attracted growing interests in recent years, and an increasing
24
number of compounds have been explored as PTT candidates. Four types of agents
25
have been reported to exhibit photothermal effects [1], including noble metal
26
nanostructures (Au [2], Pd [3]), carbon-based nanomaterials (carbon nanotubes [4],
27
graphene [5]), organic nanoparticles (NPs) like polymeric NPs [6], polypyrole NPs
28
[7]), and semiconductor nanostructures (W18O49 [8], CuS [9], Cu2-xSe [10], MoS2
29
[11] ). Among them, the noble metal nanostructures are most widely used because of
30
their relatively high photothermal conversion efficiency and low toxicity.
31
Nevertheless, the high cost of noble metal limits their broad application. In addition,
32
the main optical absorption contributed by localized plasmon resonance in noble
33
metals locates on the visible region, which brings about no benefit for in vivo
34
treatment. Compared to visible light, near-infrared (NIR) laser is much less absorbed
35
by biological tissues, and thus can penetrate several centimeters through the
36
epidermis [11]. Therefore, PTT agents with strong adsorption in the NIR (λ =
37
700-1100 nm) region and low costs are highly desired.
38
CuS, as a cheap semiconductor with strong absorption in NIR region, has been
39
extensively used for catalysis [12], chemical sensors and superionic materials in the
40
past. Recently, CuS NPs have attracted increasing interests from biomedical 3
41
researchers due to their intriguing properties. Thus, their application in PTT has been
42
explored [13,14]. Also, efforts have been devoted to fabricate CuS NPs with distinct
43
morphologies, e.g., flower like [9] and plate like [15], using various precursors and
44
methodologies. In the most of these approaches, such as hot injection, hydrothermal
45
synthesis and thermal decomposition, high temperature is required, and toxic
46
hydrogen sulfide (H2S) is released during the synthesis process. Furthermore, the
47
chemically generated nanomaterials are usually hydrophobic and exhibit a high
48
toxicity to cells when applied in biomedicine. As an alternate synthesis route, the
49
biosynthesis process relies on the reducing power of microorganisms, rather than
50
toxic organic reagents, to assemble metal ions into stable nanocrystallites. Thus,
51
microbial synthesis of NPs is environmentally friendly and cost-effective. So far,
52
numerous microorganisms including yeast, fungi and bacteria have been used as a
53
green nanofactory for producing different types of NPs, e.g., Au [16], Pd [17], CdSe
54
[18], PbS [19], etc. Although there have been a few reports on biofabricating CuS by
55
fungus [20,21], the biosynthesis of CuS NPs by bacteria has not been reported yet.
56
Shewanella oneidensis is a widely distributed Gram-negative γ-proteobacterium,
57
featured with diverse periplasmic and membrane-inserted reductases. This bacterium
58
possesses various electron pathways and secrets mediator like flavins that strengthen
59
the bioreduction of extracellular electron acceptors. It can use various terminal
60
electron acceptors including oxygen (O2), nitrate (NO3-), thiosulfate (S2O32-), ferric
61
iron (Fe3+), manganese (Mn4+) and uranium (U6+) [22]. S. oneidensis has been widely
62
studied for the bioremediation of environmental contaminants and bioenergy 4
63
generation. It is also used for the biosynthesis of sulfur-containing semiconductor NPs
64
such as As2S3 [23], ZnS [24], Ag2S [25] through reducing thiosulfate to sulfide.
65
In this work, we developed an environmentally benign method to extracellularly
66
synthesize small-sized, high stability, and well-dispersed CuS NPs by using S.
67
oneidensis MR-1 as a nanofactory under ambient temperature and pressure. Then, the
68
biofabricated CuS NPs were isolated and characterized in terms of the structure,
69
crystal phase, chemical composition and photo property. Furthermore, the
70
photothermal performance of bio-CuS NPs was also evaluated.
71 72
Materials and Methods
73 74
Biosynthesis of CuS NPs
75
S. oneidensis MR-1 was cultured in Luria-Bertani broth at 30 oC with a rotary
76
shaker (200 rpm) for 16 h until the late stationary phase. The cells were harvested by
77
centrifugation (6000×g, 4 oC, 7 min) and washed two times with sterile sodium
78
4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffered mineral
79
medium [26]. The aseptic HEPES-buffered mineral medium that was prepared under
80
anaerobic conditions by purging with pure N2 was used as the growth medium.
81
Sodium lactate (20 mM) served as the carbon source and electron donor, while
82
Na2S2O3 (1mM) was applied as the electron acceptor. The initial optical density (OD)
83
at 600 nm was about 0.1. After 24 h of growth (30 oC, 200 rpm), CuCl2 (1 mM) was
84
injected into the medium. Followed by incubation for another 24 h, the mixture was 5
85
centrifuged (8000×g, 10 min) to remove the cells, and the supernatant was
86
concentrated by ultrafiltration (10,000 Da) to collect the CuS NPs. The concentrated
87
supernatant was washed thrice by distilled water and then freeze dried for the
88
characterizations of X-ray powder diffraction (XRD), X-ray photoelectron
89
spectroscopy (XPS), and thermal stability.
90 91
Characterization
92
The morphology of the biosynthesized CuS NPs were imaged by high-resolution
93
transmission electron microscopy (HRTEM) (JEM-2000, JEOL Co., Japan) with an
94
electron kinetic energy of 200 kV. The structure of NPs was obtained from X-ray
95
powder diffraction (XRD) (TTR-III, Rigaku Co., Japan). The thermal stability of the
96
bio-CuS NPs was characterized with a DTG-60H/DSC-60 thermogravimetric analyzer
97
(Shimadzu Co., Japan) under N2 atmosphere. The heating range was from 10 oC to
98
800 oC at a rate of 10 oC per min. The chemical composition and the valence states of
99
component elements were analyzed by X-ray photoelectron spectroscopy (XPS)
100
(ESCALAB250, Thermo Fisher Inc., USA). UV-vis adsorption spectra analysis was
101
performed
102
(Shimadzu Co., Japan).
on
a
UV-3600
ultraviolet-visible-near-infrared
spectrophotometer
103 104 105 106
Photothermal property measurement To study the photothermal performance of the bio-CuS NPs, a series concentration of samples were irradiated under a 1064 nm laser (Inter-Diff Co., China) 6
107
with an output of 0.32 W for 8 min, and naturally cooled to room temperature
108
afterwards. The infrared camera was used to record the temperature variation of
109
different samples. The concentration of Cu2+ was measured by inductively coupled
110
plasma atomic emission spectroscopy (ICP-AES, PerkinElmer Inc., USA).
111 112
In vitro photothemal therapy evaluation
113
Nanoparticles tend to aggregate in vivo, thus they will be cleaned by the immune
114
system. For this reason, when nanoparticles are used for therapy or drug delivery in
115
biomedical field, surface modification is usually necessary to strengthen its
116
dispersibility. Generally polyethylene glycol (PEG) is commonly used as a modifier
117
because of its good biocompatibility and high efficiency to obstruct the aggregation of
118
nanoparticles. In order to fully simulate the practical case in the cancer therapy, a PEG
119
modification was adopted when evaluating the photothemal therapy of CuS NPs. The
120
obtained bio-CuS NPs were embellished with polyethylene glycol methelether
121
pre-modified by zinc sulfate before test. The concentration of modifier was 2 times to
122
that of the CuS NPs. After the dose of modifier, the mixture was maintained in 37 oC
123
for 24 h.
124
The in vitro photothermal therapy of the bio-CuS NPs was evaluated using the
125
methyl thiazolyl tetrazolium (MTT) assay in human pulmonary carcinoma cell lines
126
(A549R) [27]. A549R cells were seeded into a 96-well plate (5 × 103 cells per well)
127
and cultured at 37 oC and 5% CO2 for 24 h before use. Then, the culture medium was
128
removed and replaced by medium containing CuS NPs at different concentrations, 7
129
followed by further 24-h incubation. After the incubation, the cells were exposed to
130
the 1064 nm laser for 10 min (1.5 W/cm2). Subsequently, 100 µL of MTT (1 mg/mL)
131
was added to each well of the 96-well plate and reacted at 37 oC and 5% CO2 for 4 h.
132
After the reaction, 15% sodium dodecyl sulfate of 150 µL was added into the wells.
133
The assay plate was maintained overnight at room temperature, and the microplate
134
reader was applied to measure the absorbance of each well at 492 nm.
135 136 137
Results and Discussion
138 139
Biosynthesis and characteristics of the bio-CuS NPs
140
S. oneidensis accumulates S2- in the reducing medium with S2O32- as the electron
141
acceptor. After the addition of 1 mM CuCl2 and 24-h anaerobic incubation, the color
142
of the medium became brown. After centrifugation, the supernatant remained brown,
143
indicating that the CuS NPs were biosynthesized extracellularly and could be easily
144
isolated (Fig. S1). The brown-colored supernatant was collected by centrifugation and
145
characterized using TEM. The TEM image (Fig. 1a) shows that the obtained CuS NPs
146
had a uniform particle size of ~5 nm. The color of the resulting NPs was not
147
consistent with that of CuS NPs reported before (black or dark green) [28,29] , which
148
might be attributed to their tiny size. The CuS NPs showed a high solubility in water
149
with a solute concentration up to 600 µg/mL. The size of the water-dispersed CuS
150
NPs was around 14 nm, as determined by Zeta Sizer (Fig. S2). The larger size of 8
151
nanoparticles in water dispersion is attributed to the aggregation of the
152
high-concenrtation CuS NPs in solution. Microstructure information of the bio-CuS
153
nanoparticles was obtained from HRTEM. The HRTEM image (Fig. 1b) displays the
154
lattice fringe with a d-spacing of 0.304 nm, which is in agreement with the (022)
155
interplanar spacing of CuS.
156
In order to identify the phase structure of the NPs, we concentrated the
157
supernatant by ultrafiltration under anaerobic conditions. The collected samples were
158
freeze-dried for XRD analysis. The XRD pattern (Fig. 1c) of the as-synthesized
159
nanoparticles involved several characteristic peaks, such as (022), (023) and (130),
160
which were further determined to be the standard CuS phase (JCPDS card no.
161
65-7111). The XRD pattern result corresponds with the HRTEM images well.
162
Chemical composition of the bio-CuS NPs
163
Since the chemical composition of bio-synthesized NPs critically affects their
164
physical performance, thermogravimetric analysis and XPS spectra measurement
165
were conducted. The thermal degradation behavior of the bio-induced CuS NPs is
166
shown in Fig. 2. The weight loss of the sample started at 45 oC, and only 1.7% of the
167
total weight was lost below 100 oC, which could be referred to free water.
168
Subsequently, there was a major degradation peak from 98 oC to 465 oC in the
169
thermogravimetry analysis (TGA) curve, and the 16.8% weight loss suggests the
170
transformation of CuS to Cu2S. The valence states of Cu and S in the samples were
171
determined by XPS spectra. The peaks at 932.1 eV and 951.9 eV indicate Cu 2p3/2 and
172
Cu 2p1/2, respectively (Fig. 3a). In Fig. 3b, the sample displayed a slightly broad and 9
173
asymmetric peak at about 162.2 eV, while the S 2p3/2 peak position of 162.0 eV is
174
assigned to thiolate moiety (Cu-S bonds). The asymmetric XPS spectra can be fitted
175
into S 2p3/2 peak at 162.0 eV and S 2p1/2 peak at 163.5 eV. The other weaker peak
176
appeared at 168.4 eV, indicating the 2p3/2 chemical shift of a sulfonate (SO32-) [30].
177
This shift could be ascribed to the disproportionation of S2O32-. The quantification
178
analysis of XPS spectra shows the atomic ratio of Cu : S was 0.94 : 1 (Table S1),
179
which is in agreement with the ratio of CuS.
180 181
Photothermal effect of the bio-CuS NPs
182
To measure the optical property of the CuS NPs dispersed in water, the sample
183
(480 µg/mL) was analyzed by UV-vis-NIR spectroscopy. The spectrum exhibits a
184
broad-strong absorbance in the NIR region with the maximum absorption at 1100 nm
185
(Fig. 4). The peak position was different from that of the previously reported Cu2-xS
186
NPs [9,15], which might be attributed to the crystal phase diversity of individual NPs.
187
The strong NIR absorption of CuS was likely to originate from d-d intra-band
188
transitions of Cu2+, rather than the localized surface plasmon resonance. This qualifies
189
the prepared CuS a great potential in photothermal application under NIR excitation.
190
Such absorbance data inspire us to use a 1064-nm laser to test their photothermal
191
effects using a continuous irradiation with a power density of 0.53 W/cm2 for 8 min.
192
The temperature of the aqueous solution containing different concentrations (0-480
193
µg/mL) of the CuS NPs was recorded every 20 s. As shown in Fig. 5a, the samples
194
containing CuS NPs exhibited a distinguished photothermal conversion ability. With 10
195
the dose of CuS NPs (30-480 µg/mL), the temperature of CuS NPs solution increased
196
by 6-20 oC in 8 min (Fig. 5b). In contrast, the pure water exhibited a temperature
197
elevation by less than 3 oC only after the irradiation. This result demonstrates that the
198
bio-induced CuS NPs could efficiently convert the 1064 nm laser energy to thermal
199
energy.
200
The photothermal conversion efficiency of the samples was estimated using a
201
method reported by Roper et al. [31]. The conversion efficiency η of the bio-CuS NPs
202
can be calculated with Eq. (1):
203
hS (Tmax Tsur ) Q0 I (1 10 A )
(1)
204
where h is heat-transfer coefficient, S is the surface area, Tmax is the final temperature
205
after irradiation, Tsurr represents the ambient temperature, (Tmax-Tsurr) = 19.7 oC. Q0 is
206
the heat dissipated from light absorption of the solvent, which was measured
207
individually to be 11.7 mW. I is the laser power (0.53 W/cm2), and Aλ represents the
208
absorption at 1064 nm, which can be estimated to be 1.840 from Fig. 4. The value of
209
hS is derived from Eq. (2): mC hS
0 0 s 210
210
(2)
211 212
where τs is the sample system time constant, m0 and C0 represent the mass (0.3 g) and
213
specific heat capacity (4.2 J/g) of pure water, respectively.
214
Thus, the photothermal conversion efficiency of the bio-CuS NPs was calculated
215
to be 27.2%. This value is higher than that reported for Au nanorods (22%) and
216
Cu2-xSe nanocrystals (22%) [10]. 11
217
Furthermore, the photostability of the bio-CuS NPs was examined by conducting
218
four cycles of LASER ON/OFF. During each cycle, the solution of CuS NPs was
219
irradiated with the 1064 nm laser (0.53 W/cm2) for 8 min and then naturally cooled to
220
the room temperature. As shown in Fig. 7, the temperature dynamics was reproducible
221
during the cycles and showed no attenuation on the photothermal amplitude, indicating
222
that the bio-CuS NPs were highly photo-stable.
223 224
In vitro photothermal therapy of bio-CuS NPs
225
To evaluate the phtothermal therapy of the obtained CuS NPs, A549 cells were
226
incubated with the CuS NPs at different concentrations for 24 h, then exposed to 1064
227
nm laser for 10 min (1.5 W/cm2). The MTT assay was applied to determine the cell
228
viability. As shown in Fig. 8, high cell viability (> 90%) could be observed when the
229
concentration of CuS NPs was below 37.5 µg/mL without laser irradiation. However,
230
when the A549 cells were irradiated by the 1064 nm laser for 10 min, a significant
231
decrease in cell viability occurred. This results indicate that the bio-CuS NPs at a
232
relatively low concentration (37.5 µg/mL), were effective because of their high
233
photothermal conversion efficiency. This feature enables the bio-CuS to effectively kill
234
cancer cells without damaging the healthy issues, which is crucial to their biomedical
235
application.
236 237 238
Conclusions 12
239 240
In this work, small-sized (~5 nm), highly stable and hydrophilic CuS NPs with
241
distinguished photothermal conversion efficiency were successfully prepared by using
242
Shewanella oneidensis MR-1 in an eco-friendly and simple way. The biogenic CuS
243
NPs exhibited good photostability and photothermal performance with a conversion
244
efficiency up to 27.2%, higher than those of Au nanorods and Cu2-xSe nanocrystals. In
245
vitro photothermal therapy tests demonstrated the effectiveness of bio-CuS NPs,
246
ensuring bio-CuS NPs an excellent candidate for cancer treatment. In addition,
247
Shewanella oneidensis MR-1, a metal-reducing bacterium, was demonstrated to be
248
efficient in the synthesis of nanomaterials.
249 250
Acknowledgements
251
The work was supported by the Natural Science Foundation of China (21477120),
252
and the Program for Changjiang Scholars and Innovative Research Team and the
253
Collaborative Innovation Center of Suzhou Nano Science and Technology of the
254
Ministry of Education of China.
255 256
References
257 258
[1]
X. Liu, B. Li, F. Fu, K. Xu, R. Zou, Q. Wang, et al., Facile synthesis of
259
biocompatible cysteine-coated CuS nanoparticles with high photothermal
260
conversion efficiency for cancer therapy, Dalt. Trans. 43 (2014) 11709–11715. 13
261
[2]
L. Au, D. Zheng, F. Zhou, Z.-Y. Li, X. Li, Y. Xia, A quantitative study on the
262
photothermal effect of immuno gold nanocages targeted to breast cancer cells,
263
ACS Nano. 2 (2008) 1645–1652.
264
[3]
W. Fang, S. Tang, P. Liu, X. Fang, J. Gong, N. Zheng, Pd Nanosheet‐
265
Covered Hollow Mesoporous Silica nanoparticles as a platform for the chemo
266
‐photothermal treatment of cancer cells, Small. 8 (2012) 3816–3822.
267
[4]
H.K. Moon, S.H. Lee, H.C. Choi, In vivo near-infrared mediated tumor
268
destruction by photothermal effect of carbon nanotubes, ACS Nano. 3 (2009)
269
3707–3713.
270
[5]
K. Yang, L. Hu, X. Ma, S. Ye, L. Cheng, X. Shi, et al., Multimodal imaging
271
guided photothermal therapy using functionalized graphene nanosheets
272
anchored with magnetic nanoparticles, Adv. Mater. 24 (2012) 1868–1872.
273
[6]
H. Gong, L. Cheng, J. Xiang, H. Xu, L. Feng, X. Shi, et al., Near‐infrared
274
absorbing polymeric nanoparticles as a versatile drug carrier for cancer
275
combination therapy, Adv. Funct. Mater. 23 (2013) 6059–6067.
276
[7]
Z. Zha, X. Yue, Q. Ren, Z. Dai, Uniform polypyrrole nanoparticles with high
277
photothermal conversion efficiency for photothermal ablation of cancer cells,
278
Adv. Mater. 25 (2013) 777–782.
279
[8]
Z. Chen, Q. Wang, H. Wang, L. Zhang, G. Song, L. Song, et al., Ultrathin
280
PEGylated W18O49 nanowires as a new 980 nm‐laser‐driven photothermal
281
agent for efficient ablation of cancer cells in vivo, Adv. Mater. 25 (2013)
282
2095–2100. 14
283
[9]
Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, et al., Hydrophilic flower
284
‐like CuS superstructures as an efficient 980 nm laser‐driven photothermal
285
agent for ablation of cancer cells, Adv. Mater. 23 (2011) 3542–3547.
286
[10] C.M. Hessel, V. P. Pattani, M. Rasch, M.G. Panthani, B. Koo, J.W. Tunnell, et
287
al., Copper selenide nanocrystals for photothermal therapy, Nano Lett. 11
288
(2011) 2560–2566.
289
[11] S. Wang, K. Li, Y. Chen, H. Chen, M. Ma, J. Feng, et al., Biocompatible
290
PEGylated MoS2 nanosheets: Controllable bottom-up synthesis and highly
291
efficient photothermal regression of tumor, Biomaterials. 39 (2015) 206–217.
292
[12] F. Li, J. Wu, Q. Qin, Z. Li, X. Huang, Controllable synthesis, optical and
293
photocatalytic properties of CuS nanomaterials with hierarchical structures,
294
Powder Technol. 198 (2010) 267–274.
295
[13] M. Zhou, R. Zhang, M. Huang, W. Lu, S. Song, M.P. Melancon, et al., A multifunctional
[64Cu]
296
chelator-free
CuS
nanoparticle
platform
for
297
simultaneous micro-PET/CT imaging and photothermal ablation therapy, J. Am.
298
Chem. Soc. 132 (2010) 15351–15358.
299
[14] Z. Zha, S. Wang, S. Zhang, E. Qu, H. Ke, J. Wang, et al., Targeted delivery of
300
CuS nanoparticles through ultrasound image-guided microbubble destruction
301
for efficient photothermal therapy, Nanoscale. 5 (2013) 3216–3219.
302
[15] Q. Tian, F. Jiang, R. Zou, Q. Liu, Z. Chen, M. Zhu, et al., Hydrophilic Cu9S5
303
nanocrystals: A photothermal agent with a 25.7 % heat conversion efficiency
304
for photothermal ablation of cancer cells in vivo, ACS Nano. 5 (2011) 15
305
9761–9771.
306
[16] A.K. Suresh, D.A. Pelletier, W. Wang, M.L. Broich, J.-W. Moon, B. Gu, et al.,
307
Biofabrication of discrete spherical gold nanoparticles using the metal-reducing
308
bacterium Shewanella oneidensis, Acta Biomater. 7 (2011) 2148–2152.
309
[17] L. Jia, Q. Zhang, Q. Li, H. Song, The biosynthesis of palladium nanoparticles
310
by antioxidants in Gardenia jasminoides Ellis: long lifetime nanocatalysts for
311
p-nitrotoluene hydrogenation, Nanotechnology. 20 (2009) 385601.
312
[18] R. Cui, H. Liu, H. Xie, Z. Zhang, Y. Yang, D. Pang, et al., Living yeast cells as
313
a controllable biosynthesizer for fluorescent quantum dots, Adv. Funct. Mater.
314
19 (2009) 2359–2364.
315
[19] S. Seshadri, K. Saranya, M. Kowshik, Green synthesis of lead sulfide
316
nanoparticles by the lead resistant marine yeast, Rhodosporidium diobovatum,
317
Biotechnol. Prog. 27 (2011) 1464–1469.
318
[20] M. Schaffie, M.R. Hosseini, Biological process for synthesis of semiconductor
319
copper sulfide nanoparticle from mine wastewaters, J. Environ. Chem. Eng. 2
320
(2014) 386–391.
321
[21] M.R. Hosseini, M. Schaffie, M. Pazouki, E. Darezereshki, M. Ranjbar,
322
Biologically synthesized copper sulfide nanoparticles: Production and
323
characterization, Mater. Sci. Semicond. Process. 15 (2012) 222–225.
324
[22] T. Perez-Gonzalez, C. Jimenez-Lopez, A.L. Neal, F. Rull-Perez, A.
325
Rodriguez-Navarro, A. Fernandez-Vivas, et al., Magnetite biomineralization
326
induced by Shewanella oneidensis, Geochim. Cosmochim. Acta. 74 (2010) 16
327
967–979.
328
[23] J.-H. Lee, M.-G. Kim, B. Yoo, N. V Myung, J. Maeng, T. Lee, et al., Biogenic
329
formation of photoactive arsenic-sulfide nanotubes by Shewanella sp. strain
330
HN-41, Proc. Natl. Acad. Sci. 104 (2007) 20410–20415.
331
[24] X. Xiao, X.-B. Ma, H. Yuan, P.-C. Liu, Y.-B. Lei, H. Xu, et al., Photocatalytic
332
properties of zinc sulfide nanocrystals biofabricated by metal-reducing
333
bacterium Shewanella oneidensis MR-1, J. Hazard. Mater. 288 (2015)
334
134–139.
335
[25] A.K. Suresh, M.J. Doktycz, W. Wang, J.-W. Moon, B. Gu, H.M. Meyer, et al.,
336
Monodispersed biocompatible silver sulfide nanoparticles: Facile extracellular
337
biosynthesis using the γ-proteobacterium, Shewanella oneidensis, Acta
338
Biomater. 7 (2011) 4253–4258.
339
[26] D.-B. Li, Y.-Y. Cheng, C. Wu, W.-W. Li, N. Li, Z.-C. Yang, et al., Selenite
340
reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in
341
periplasm, Sci. Rep. 4 (2014).
342
[27] Y. Min, C. Mao, S. Chen, G. Ma, J. Wang, Y. Liu, Combating the drug
343
resistance of cisplatin using a platinum prodrug based delivery system, Angew.
344
Chemie Int. Ed. 51 (2012) 6742–6747.
345
[28] W. Feng, W. Nie, Y. Cheng, X. Zhou, L. Chen, K. Qiu, et al., In vitro and in
346
vivo toxicity studies of copper sulfide nanoplates for potential photothermal
347
applications, Nanomedicine Nanotechnology, Biol. Med. 11 (2015) 901–912.
348
[29] C. Zhang, Y.-Y. Fu, X. Zhang, C. Yu, Y. Zhao, S.-K. Sun, BSA-directed 17
349
synthesis of CuS nanoparticles as a biocompatible photothermal agent for
350
tumor ablation in vivo, Dalt. Trans. 44 (2015) 13112–13118.
351
[30] T. Nakanishi, B. Ohtani, K. Uosaki, Fabrication and characterization of
352
CdS-nanoparticle mono-and multilayers on a self-assembled monolayer of
353
alkanedithiols on gold, J. Phys. Chem. B. 102 (1998) 1571–1577.
354
[31] D.K. Roper, W. Ahn, M. Hoepfner, Microscale heat transfer transduced by
355
surface plasmon resonant gold nanoparticles, J. Phys. Chem. C. 111 (2007)
356
3636–3641.
357
18
Figure captions
Figure 1. (a) Low-magnification; (b) HRTEM image; and (c) XRD patterns of the bio-CuS NPs
Figure 2. Thermogravimetric analysis (TGA) of the bio-CuS NPs
Figure 3. XPS spectra of (a) Cu 2p and (b) S 2p regions of the bio-CuS NPs
Figure 4. UV-vis absorbance spectra of the bio-CuS NPs dispersed in water
Figure 5. (a) Temperature elevation of pure water and the solution of CuS NPs at different concentrations of CuS (30, 60, 120, 240, 480 µg/mL) under the irradiation of 1064 nm for 480 s; (b) plot of temperature change (ΔT) over a time period of 480 s versus the CuS concentration
Figure 6. (a) Infrared images of the CuS NPs solution (480 µg/mL) irradiated by NIR laser (1064 nm, 8 min), and then shut off; (b) temperature dynamics of the CuS NPs solution; and (c) time constant of heat transfer for the system was determined to be 161.5 s by applying the time data of cooling period vs the negative natural logarithm of driving force temperature (
T Tsurr ). Tmax Tsurr
Figure 7. Temperature elevation of the bio-CuS NPs over four LASER ON/OFF cycles
Figure 8. Cell viability of A549 cells after treatment by bio-CuS NPs at different concentrations and with different irradiation times
19
Figure 1. (a) Low-magnification; (b) HRTEM image; and (c) XRD patterns of the bio-CuS NPs
20
Figure 2. Thermogravimetric analysis (TGA) of the bio-CuS NPs
21
Figure 3. XPS spectra of (a) Cu 2p and (b) S 2p regions of the bio-CuS NPs
22
Figure 4. UV-vis absorbance spectra of the bio-CuS NPs dispersed in water
23
Figure 5. (a) Temperature elevation of pure water and the solution of CuS NPs at different concentrations of CuS (30, 60, 120, 240, 480 µg/mL) under the irradiation of 1064 nm for 480 s; (b) plot of temperature change (ΔT) over a time period of 480 s versus the CuS concentration
24
Figure 6. (a) Infrared images of the CuS NPs solution (480 µg/mL) irradiated by NIR laser (1064 nm, 8 min), and then shut off; (b) temperature dynamics of the CuS NPs solution; and (c) time constant of heat transfer for the system was determined to be 161.5 s by applying the time data of cooling period vs the negative natural logarithm of driving force temperature (
T Tsurr ). Tmax Tsurr
25
Figure 7. Temperature elevation of the bio-CuS NPs over four LASER ON/OFF cycles
26
Figure 8. Cell viability of A549 cells after treatment by bio-CuS NPs at different concentrations and with different irradiation times
27