Materials Science and Engineering C 68 (2016) 798–804

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Materials Science and Engineering C

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

One-step synthesis of soy protein/graphene nanocomposites and their

application in photothermal therapy
Xuejiao Jiang, Zhao Li, Jinrong Yao, Zhengzhong Shao, Xin Chen ⁎
State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Polymers and Polymer Composite Materials, Department of Macromolecular Science, Laboratory of
Advanced Materials, Fudan University, Shanghai 200433, People's Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Photothermal therapy, due to its security and effectiveness, has recently become a promising cancer treatment
Received 12 April 2016 after surgery, radiotherapy, chemotherapy, and biological therapy. In this article, soy protein isolate/reduced
Received in revised form 4 July 2016 graphene oxide (SPI/rGO) nanocomposites are prepared via a simple and green process. That is, GO is reduced
Accepted 16 July 2016
in situ and stabilized by SPI, an abundant, low-cost, sustainable natural material, and simultaneously forms
Available online 18 July 2016
SPI/rGO nanocomposites. The resulting SPI/rGO nanocomposites disperse in water very well and exhibit good
Keywords:
biocompatibility due to the attachment of SPI to the surface of rGO. Such SPI/rGO nanocomposites demonstrate
Plant proteins an excellent photothermal capacity and are able to kill HeLa cells efficiently with near-infrared irradiation
Carbon materials (808 nm). The results in this work suggest that such a SPI/rGO hybrid material could be a promising candidate
Hybrid materials for future applications of photothermal therapy.
Photothermal transformation © 2016 Elsevier B.V. All rights reserved.
Biocompatibility

1. Introduction
In the past quarter century, although there were many explorations
in cancer biology, such studies have not been translated into even
remotely comparable advances in clinical practice [1,2]. Current cancer
treatments still primarily focus on traditional surgical intervention,
radiation and chemotherapeutic drugs, which often damage healthy
cells and tissues, or even cause toxicity in patients. However, a new
and non-invasive modality called photothermal therapy (PTT) has
been increasingly recognized as a promising alternative to traditional
cancer therapies, such as surgery, radiotherapy, and chemotherapy,
due to its spatiotemporal selectivity and minimal invasiveness [3,4].
PTT usually utilizes photo-absorbing agents, including graphene oxide
(GO) [5,6], carbon nanotubes [7,8], gold nanoparticles [9–12], and or-
ganic near-infrared (NIR) dyes [6,13] (such as cyanine) to generate a
high localized temperature for ablating tumors. This advantage allows
clinicians to save most of the healthy tissues close to the targeted lesions
by using near infrared (NIR) light irradiation [13,14].
Graphene, which is one atom thick and has a two-dimensional (2D)
single carbon layer, is emerging as a rising star in material science and
condensed matter physics [15] due to its high specific surface area and
excellent mechanical, electrical, and thermal properties [16–18]. These
properties enable graphene to be considered an ideal material for a
variety of applications. Recently, Yang [19], Kuilla [20], Meng [21] and
their coworkers have explored the photothermal effect of graphene in
⁎ Corresponding author.
E-mail address: chenx@fudan.edu.cn (X. Chen).
polymer composites. They found that graphene had the potential to
convert light to heat efficiently and that the conversion was even better
than carbon nanotubes when graphene was used as a nanoscale heater
and energy-transfer unit for infrared (IR)-triggered actuators. The
perfect sp2 carbon-network structure of graphene ensures that it
efficiently absorbs and transforms IR light into thermal energy [22].
However, the potential of graphene in bionanotechnology is somehow
underestimated due to its disadvantages, such as highly hydrophobic
and featureless surfaces, low aqueous solubility, and poor biocompatibil-
ity [23]. To improve these drawbacks, several natural biomacromolecules
(DNAs and proteins) have been applied to modify the surface of graphene
nanosheets [24–26]. In our previous research, we demonstrated that silk
fibroin was able to grow on the surface of graphene nanosheets in the
form of nanofibrils by self-assembly. The graphene/silk nanofibril hybrid
preserved the high electrical conductivity of pure graphene. In addition,
the graphene/silk nanofibril hybrid film prepared via a vacuum-assisted
filtering process showed good flexibility and biocompatibility [23,27].
Soy protein isolate (SPI), which is one of the most abundant natural
polymers extracted from soybeans, has attracted comprehensive inter-
ests in the field of material science [28] due to its sustainability, low
cost, functionality, biocompatibility, and tunable degradability. There
have been several soy protein-based materials, such as plastics [29],
gels [30], films [31,32], and biomedical materials [33] reported in the
literature.
The synthesis of SPI/inorganic composites in previous research has
mainly focused on improving the mechanical performance of SPI mate-
rials by using inorganic materials as strengthening agents. For example,
Lee and his coworkers prepared SPI/montmorillonite (SPI/MMT)

http://dx.doi.org/10.1016/j.msec.2016.07.034
0928-4931/© 2016 Elsevier B.V. All rights reserved.
 X. Jiang et al. / Materials Science and Engineering C 68 (2016) 798–804
composite film with a melt extrusion method for drug release [34]. How-
ever, the preparation conditions were relatively harsh, and the structure
of the obtained composite was not homogeneous at the nanoscale.
Zhuang and his coworkers have reported the preparation of SPI/
graphene aerogel with a hydrothermal method, and the obtained com-
posite was used as an adsorbent to remove antibiotics (ciprofloxacin
and tetracycline) from aqueous solutions [35,36]. They found the SPI/
graphene composites showed better hydrophilicity, improved biologi-
cal compatibility, and lower cytotoxicity compared with pure graphene.
During their preparation, they need add ascorbic acid as a reductant,
and the application of such a SPI/graphene composite was for water
treatment.
SPI is rich with sulfhydryl groups, and it also contains reducing
amino acid residues such as tyrosine. Therefore, SPI may be able to
reduce GO to prepare graphene, which we call reduced GO, i.e., rGO.
In addition, SPI is an amphiphilic polymer (a nature for all proteins),
which makes it well known for its adhesiveness to solid surfaces [37].
There are already several reports indicating that other proteins, such
as bovine serum albumin (BSA) and silk fibroin, are able to attach to
the surface of graphene by hydrophobic interaction and π-π stacking
[24]. Thus, SPI may have the potential to reduce GO in situ and then
attach to the surface of the resulting rGO nanosheets to ultimately
form a hybrid nanocomposite.
In this article, we show evidence that we were able to obtain a SPI/
rGO nanocomposite via a facile, mild, and one-step protocol. SPI was
used as both the reducing and stabilizing agent, and it improved the
biocompatibility of the resulting SPI/rGO nanocomposite. Then we
demonstrated that a SPI/rGO nanocomposite could be a promising
photothermal agent for cancer therapy.
2. Materials and methods
2.1. Materials
SPI powder (protein content N90%) was obtained from Shenyuan
Food Co. Ltd., Shanghai, China. Dithiothreitol (99%) and 5,5′-Dithiobis-
(2-nitrobenzoic acid) (DTNB) were purchased from Aladdin, Shanghai,
China. Graphite powder, concentrated H2SO4 (98%), KMnO4, NaOH
and guanidine hydrochloride (99%) were purchased from Sinopharm
Chemical Reagent Co., Ltd., Shanghai, China. All chemicals used in this
work were analytical grade and used without further purification.
The preparation of an SPI aqueous solution followed a well-
established procedure, as reported in our previous work [38]. Briefly,
SPI powder was dissolved in aqueous solution containing 6 mol/L guani-
dine hydrochloride and 1 mmol/L dithiothreitol. After dialysis against
NaOH aqueous solution (pH = 11.5) for two days and deionized
water for another day at room temperature, the SPI solution was obtain-
ed. The resulting SPI solution was transferred into the freezer compart-
ment of a refrigerator and then freeze-dried to obtain pure SPI powder.
GO was synthesized using a modified Hummers method [39]. Brief-
ly, 3.7 g of graphite power were mixed into 120 mL concentrated
sulphuric acid (98.5%) that was pre-cooled by an ice bath. Next, 2.5 g
NaNO3 and 11.6 g KMnO4 were slowly added under continuous vigor-
ous stirring such that the temperature was constantly maintained
below 20 °C. The mixture was then stirred continuously at 35 °C for
7 h. After 9.5 g KMnO4 was added, the reaction was left running uninter-
rupted at 35 °C for 14 h. The reaction was quenched by slowly pouring
the mixture into 600 mL H2O under stirring. Finally, 20 mL of 30%
H2O2 was added, and the mixture turned bright yellow and bubbled.
The mixture was filtered and washed with 1 L 5% HCl aqueous solution
and 200 mL ethanol. The solid powder was dried at 70 °C.
2.2. Preparation and characterization of SPI/rGO nanocomposites
Desired quantities of SPI powder were added to the GO solutions
under vigorous stirring to prepare a final SPI/GO mixture (10 mL)
799
with a GO concentration of 0.1 wt% and solid mass ratios of 10:1, 50:1,
100:1 for SPI/GO. These samples were named SG10, SG50, and SG100,
respectively, for both the SPI/GO mixtures and final SPI/rGO nanocom-
posites. Subsequently, the SPI/GO mixtures were incubated at 70 °C
for 24 h to obtain SPI/rGO nanocomposites.
TEM images of the synthesized nanocomposites were observed with
an FEI Tecnai G2 20 TWIN transmission electron microscope at 200 kV.
The samples were prepared by drying the diluted nanocomposite
solutions on a formvar/carbon-coated copper grid. AFM images were ac-
quired using a Multi-mode 8 atomic force microscope in tapping mode.
X-ray diffraction (XRD) data were recorded on an X'pert Pro X-ray
diffractometer with Cu Kα radiation. Raman spectra were obtained
with a Renishaw inVia Reflex spectrometer coupled to a Lieca micro-
scope at a wavelength of 785 nm for a He-Ne laser. The energy of the
laser was 0.6 mW, which is only 1/10 that used for a normal laser,
because the graphene would be carbonized under high energy.
2.3. Measurement of free sulfhydryl with the Ellman method [40]
Ellman's reagent (10 mM DTNB) was prepared by suspending
198.2 mg DTNB in 50 mL of 50 mmol/L Na2HPO4 solution, and all
DTNB was dissolved. Aliquots of all reagents were immediately frozen
at −25 °C, and thawed portions were used within 1 day. Next, 100 μL
1 wt% of SG10 and SPI samples was diluted with Tris-HCl buffer
(pH = 8) to a final volume of 1 mL. After adding 100 μL of 10 mmol/L
Ellman reagent, the samples were immediately vortexed, and the A412
values were read against the samples using 100 μL Tris-HCl buffer
instead of Ellman's regent as a blank after a 10-min incubation time
(1 cm light path).
The amount of free sulfhydryl in the SPI sample was calculated as
follows:


½SH ðμmol=g soy proteinÞ ¼ 1; 000; 000  ½ASPI −½ASG10 =ðε  b  cÞ
where the standard value ε412 = 14,150 L/mol·cm, b = 1 cm, and c
refers to the concentration of SPI (g/L)
2.4. Cytotoxicity tests
The cytotoxicity of GO and SPI/rGO nanocomposites was determined
by a CCK-8 assay on L929 cells. L929 cells were plated in a 96-well plate
at a seeding density of 10,000 cells/well with 100 μL of DMEM media at
various concentrations of GO and SPI/rGO. After incubating for 24 h at
37 °C, the solutions were removed from the well plates immediately
prior to the addition of a colorimetric indicator. PBS was used to wash
the residual nanocomposites to prevent any interference in the absor-
bance readings.
2.5. In vitro photothermal performance and photothermal therapy (PTT)
effects on HeLa cells
The SPI/rGO nanocomposite solution samples were diluted to a
desired concentration of 100 μg/mL with distilled water. Then, 1.2 mL
SPI/rGO nanocomposite solution samples (i.e., SG10, SG50 and
SG100), pure GO solution (100 μg/mL), pure SPI solution (0.1 wt%),
and deionized water were continuously irradiated by an NIR laser
(808 nm, 2.5 W) at a distance of 5 cm for 5 min in 1.5 mL vials. The
photostability of the SPI/rGO nanocomposite samples was assessed by
switching the laser on/off five times. Temperature was measured by
an infrared thermal camera. All experiments were conducted at room
temperature (~20 °C).
The PTT effect of the SPI/rGO nanocomposite was determined by a
CCK-8 assay on HeLa cells. Digested HeLa cells were mixed with a
desired amount of pure GO, SG10, SG50 and SG100 solution in 1.5 mL
vials. The concentrations of graphene in each vial were set as 40, 60,
80, and 100 μg/mL, respectively. After 1 h of incubation, these HeLa
800 X. Jiang et al. / Materials Science and Engineering C 68 (2016) 798–804
Fig. 1. Digital photos of solutions containing (a) pure GO, (b) SG10, (c) SG50, (d) SG100.
cell solutions were then irradiated for 3 min with a 2.5 W 808 nm diode
laser. Immediately after heating, HeLa cells were plated in a 96-well
plate with a cell density of 10,000 cells/well. After 24 h of incubation
at 37 °C, the cell viability was determined by a CCK-8 assay using the
same protocol as the cytotoxicity test shown above.
3. Results and discussion
3.1. Preparation and characterization of SPI/rGO nanocomposites
To prepare the SPI/rGO nanocomposites we designed, we first added
a certain amount of SPI powder to 0.1 wt% GO solution and then incu-
bated the solution at 70 °C under continuous stirring for 24 h. We
found the resulting solution was very stable, which was almost the
same as pure GO solution (Fig. 1). In addition, these solutions were
able to be stored under 4 °C for several months without any aggregation.
Although these solutions have the same appearance, the TEM im-
ages of the surface morphology of the GO nanosheets were different
(Fig. 2). Fig. 2a shows that the surface of the GO nanosheet was smooth.
Fig. 2. TEM images of (a) pure GO, (b) SG10, (c) SG50, and (d) SG100.
However, after adding SPI to the GO solution and incubating it for a
period of time, the surface of the GO nanosheet became rough. The
more SPI that was added, the rougher the GO surface was (Fig. 2b–d).
This phenomenon clearly indicates that globular soy proteins were
assembled on the surface of the GO nanosheets.
In contrast, AFM could be used not only to observe the surface mor-
phology of GO nanosheet but also to estimate the average thickness of
the GO nanosheets before and after the addition of SPI. Fig. 3a shows
that the thickness of pure GO was b1 nm, which was consistent with
the height of mono-layer GO. However, the thickness of GO after adding
SPI (for example, SG50 in Fig. 3b) significantly increased to 10–15 nm,
which further confirmed the attachment of soy protein on the surface
of the GO nanosheet.
Although we confirmed that soy proteins were successfully attached
to the surface of the GO nanosheet, we still did not know whether the
soy protein changed the structure of GO. Therefore, X-ray diffraction
(XRD) and Raman spectroscopy were used to characterize the structural
details of the nanocomposite. The XRD pattern of pure soy protein
showed a main crystalline peak at approximately 20° and a small peak
at approximately 10° (Fig. 4a, curve a), which correlates with those
reported in the literature [41,42]. In the meantime, pure GO showed a
strong peak at 10.97° (curve b), which corresponded to the typical
diffraction peak of the (001) plane in the GO nanosheet [43]. However,
the typical diffraction peak of GO at 10.97° disappeared after the
addition of SPI, and the nanocomposite only showed a broad peak at
approximately 20° (curve c–e) that was similar to pure SPI. Such a result
demonstrated that the GO nanosheets were exfoliated and provided
additional evidence for the successful separation of graphene in the
form of a mono-layer [20,44] after the addition of SPI.
Meanwhile, the Raman spectra shown in Fig. 4b suggest that GO
was reduced by SPI. The pure GO and nanocomposites showed strong
twin peaks at 1308 and 1583 cm−1. It is well known that the peak
 X. Jiang et al. / Materials Science and Engineering C 68 (2016) 798–804
Fig. 3. AFM images and height profiles for (a) pure GO and (b) SG50.
at 1308 cm− 1 is assigned to the D band (associated with the order/
disorder degree of the system), which is attributed to the breathing
mode of the k-point phonons of A1g symmetry. The peak at 1583 cm−1
is assigned to the G band (an indicator of the stacking structure),
which results from the E2g phonon of sp2 carbon atoms [45]. Thus, the
D/G intensity ratio is defined as a parameter for measuring the sp3 do-
main percentage of a carbon structure containing both sp3 and sp2
bonds [46] and the degree of exfoliation/disorder in graphene material
[45]. Previous studies indicate that when GO is chemically reduced to
rGO, the surface defects and the carbon bond fractures are increased,
and the topological disorder is introduced in graphene structure,
resulting in the increase of D/G intensity ratio. The higher the D/G inten-
sity ratio is observed, the more the GO is reduced to rGO [47,48]. From
Fig. 4b, we were able to calculate the D/G intensity ratios of pure GO,
SG10, SG50, SG100 as 1.29, 1.83, 2.03, and 2.30, respectively. It is clear
that the D/G intensity ratio of the nanocomposite was larger than that
of pure GO and increased as the amount of added SPI increased. There-
fore, we are confident that GO has been partially reduced by SPI, and
the more SPI was added, the more GO was reduced. Additionally, the
Raman intensity of SPI in our experimental conditions was negligible
compared to that of the graphene materials, so it had little effect on the
D/G intensity ratio.
SPI contains reducing amino acid residues, such as tyrosine and cys-
teine [34]. Previous studies have already confirmed that the phenolic
Fig. 4. XRD patterns (a) and Raman spectra (b) of pure SPI, pure GO, and the
corresponding nanocomposites. (I) pure SPI; (II) pure GO; (III) SG10; (IV) SG 50; (V)
SG100.
801
group in tyrosine has an electron donating ability that is sufficiently
strong to reduce the noble metal to nanoparticles [49] and GO to rGO
[37]. Therefore, there is no doubt that tyrosine in SPI was also able to
reduce GO in the current system. On the other hand, when we prepared
an SPI aqueous solution, we used dithiothreitol to destroy the disulfide
bond generate abundant free sulfhydryl groups. Monahan and his
coworkers [50] reported that the oxidization of sulfhydryl occurred
when the temperature increased to 70 °C or above in neutral solution,
which demonstrated that the sulfhydryl groups had reductive proper-
ties. Therefore, we have reason to believe that the sulfhydryl groups in
SPI also contributed to the reduction process in our study.
To prove whether the sulfhydryl groups in SPI also served as a reduc-
ing agent to reduce GO, the Ellman method was used to measure the
amount of free sulfhydryl in SPI and SG10. Fig. 5 shows that the SPI
solution had a sulfhydryl characteristic peak at 412 nm, which almost
disappeared in the SG10 sample and implies that there was a significant
decrease in free sulfhydryl after forming the nanocomposite of SPI and
GO. We were able to calculate that the reaction amount of free sulfhy-
dryl in our pure SPI sample was 36.7 μmol/g soy protein, which is
reasonable because the theoretical sulfhydryl amount was 90 μmol/g
soy protein, according to the cysteine percentage in the 7S and 11S
components of SPI [34]. These results confirmed that there were free
sulfhydryl groups in our pure SPI sample and that the reducing sulfhy-
dryl groups were consumed after they reacted with GO and reduced
GO to rGO. These characterizations show that we successfully prepared
a SPI/rGO nanocomposite.
3.2. Cytotoxicity tests
Biocompatibility is the one of the most important aspects when de-
veloping a biomaterial. We know that SPI has good biocompatibility
[49], but GO remains somewhat cytotoxicity [51] and rGO even has
more cytotoxicity [52]. Therefore, we used SG10 as an example to inves-
tigate the cytotoxicity because it contains less SPI than SG50 and SG100.
Fig. 6 shows the cytotoxicity of GO and SG10 on L929 cells by measuring
their relative cell viabilities after 24 h of incubation through a CCK-8
assay. We can see that at low graphene concentrations, neither pure
GO nor SG10 show obvious cytotoxicity, and there was no significant
difference between them. However, with an increase in the graphene
concentration to 50 μg/mL, significant differences appeared in the cell
viability between pure GO and SG10. When the graphene concentration
increased to 250 μg/mL, the cell viability for pure GO dropped to b 80%
and exhibited some cytotoxicity. In contrast, the cell viabilities of SG10
remained very high for all graphene concentrations that were tested,
even higher than 100%, because the soy protein itself may be considered
an additional nutrilite for L929 cells. Therefore, the cytotoxicity result
implies that the SPI/rGO nanocomposite is more biocompatible than
pure GO.
Fig. 5. UV–vis spectra of (a) pure SPI and (b) SG10.
802 X. Jiang et al. / Materials Science and Engineering C 68 (2016) 798–804
Fig. 6. Cytotoxicity of pure GO and SG10 in L929 cells after incubation for 24 h. Data were
expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-
way analysis of variance (ANOVA), * denotes p b 0.05.
3.3. In vitro photothermal heating characterization and photothermal
therapy effects on HeLa cells
To verify the potential of the SPI/rGO nanocomposite as a
photothermal agent, the photothermal effect of the composites was
evaluated by measuring the temperature change of the solutions after
they were exposed to an 808 nm laser (2.5 W, 5 min). Fig. 7 shows
the visual photothermal images of the vials containing pure GO and
different SPI/rGO nanocomposites. The image of the SPI/rGO nanocom-
posite was brighter than that of pure GO, and sample SG100 was the
brightest. Such a phenomenon is easy to be understood because it is
well known that the photothermal performance of GO is much worse
than rGO [53]. The temperature increase curves are also shown in
Fig. 8. With the increase in irradiation time, the temperature increased
faster with an increasing amount of SPI added when the nanocompos-
ites formed. The temperature for the SG10, SG50, and SG100 solutions
reached approximately 40, 70, and 80 °C, respectively, from room
temperature at approximately 20 °C. In contrast, under the same laser
irradiation conditions, the temperature curve for 100 μg/mL of pure
SPI solution was similar to that of pure water without any obvious
temperature changes. In the meantime, pure GO showed a weak
photothermal effect with a temperature increase b 10 °C. Therefore,
the significant photothermal effect of the SPI/rGO nanocomposite
Fig. 7. NIR imaging of pure GO (a), SG10 (b), SG50 (c) and SG100 (d) solution
temperatures after exposure to an 808 nm laser. Each image shows the surface
temperature of the vial. The temperature scale bar is between 20 and 80 °C. The
concentration of graphene was 100 μg/mL.
Fig. 8. Measurement of sample temperature changes versus time during 808 nm NIR
irradiation (2.5 W, 8 min). The concentration of graphene was 100 μg/mL.
could be attributed to the successful reduction of GO by SPI. It is clear
that as more SPI was added, more GO was reduced. On the other
hand, more SPI aggregates in the nanocomposite solution may also
have scattered more NIR light, which probably also contributed to the
photothermal effect [54].
There are several reports regarding the photothermal transforma-
tion properties of graphene-based materials in the literature. However,
it is not easy to compare their properties directly with ours because the
experimental conditions that carried out in different research groups
were varied. The volume of the solution to be irradiated, the concentra-
tion of photothermal agent in the solution, the irradiation time, the
power and even the wavelength of the laser may be different [55–60].
Generally, if we compare the increase of the temperature after 5 min
of irradiation, such a value was from 3 to 42 °C for these graphene-
based materials reported. Among them, the most similar system to
ours is BSA/rGO, but its temperature increase was 30 °C [55] or 40 °C
[56], which is lower than our SPI/rGO system. In addition, we shall
emphasis that we did not introduce any reducing agent, like hydrazine
hydrate in our preparation process, which may greatly improve the
biocompatibility and environmental friendliness in our SPI/rGO
nanocomposites.
We also tested the photothermal stability of SPI/rGO nanocompos-
ites by switching the laser on/off for five cycles. The temperature
increased when the laser was on and decreased to room temperature
when the laser was turned off. The increase/decrease curve of the
temperature showed a very good repeatability at least for five cycles
(Figures not shown). This outcome indicates that the SPI/rGO nanocom-
posite presented an excellent photothermal capability and stability
under repeated laser irradiation, which makes it a promising candidate
for a PTT agent.
Finally, we evaluated the feasibility of using the SPI/rGO nanocom-
posite as a photothermal agent for cancer therapy. The in vitro PTT effect
of the SPI/rGO nanocomposite was studied in HeLa cells under 808 nm
laser irradiation. Fig. 9 shows all SPI/rGO nanocomposites killed the
HeLa cells more efficiently than pure GO under NIR irradiation. For ex-
ample, at the lowest graphene concentration (40 μg/mL), pure GO
only killed b20% of the HeLa cells, but the worst SPI/rGO composite
(sample SG10) killed N50% of the cells, and the best composite (sample
SG100) killed almost 80% of the HeLa cells. With an increase in graphene
concentration, the difference between pure GO and SPI/rGO becomes
more significant. When the graphene concentration increased to
80 μg/mL, all SPI/rGO nanocomposites killed N95% of the HeLa cells.
This outcome coincides with the photothermal heating characterization
results. Interestingly, the SPI/rGO nanocomposites also exhibited an SPI
dependent cytotoxicity to the HeLa cells that correlated with the
photothermal test results. At relatively low graphene concentrations
(40 and 60 μg/mL), the cell viability showed a significant difference
among SG10, SG50, and SG100, that is SG100 can kill most HeLa cells,
whereas SG10 showed the worst cell toxicity. It is possible that with
 X. Jiang et al. / Materials Science and Engineering C 68 (2016) 798–804
Fig. 9. Photothermal therapy effects on HeLa cells with pure GO, SG10, SG50, and SG100.
Data are expressed as mean ± standard deviation (SD). Statistical analysis was
performed with one-way analysis of variance (ANOVA), * denotes p b 0.05, ** denotes
p b 0.01, *** denotes p b 0.001.
the increase in graphene concentrations, even SG10 can generate
enough heat to kill HeLa cells. As such, there was no obvious difference
among SG10, SG50, and SG100. In summary, SG 100 showed the best
PPT effect in HeLa cells with an effective concentration of 60 μg/mL,
but SG10 and SG50 needed a higher effective concentration of 80 μg/mL.
It should be noted that the PTT still has some drawbacks. The main
possible side effect of photothermal agents in PTT is that, while the
high temperature kills cancer cells, it increases the risk of damaging
the normal tissues near the lesions through heat conduction, which
may affect the therapeutic accuracy [61].
4. Conclusions
In this study, we successfully prepared SPI/rGO nanocomposites
through an easy, green, and one-step approach. We demonstrated that
SPI molecules attached to the surface of the GO nanosheets and reduced
GO to rGO in situ with various characterizations, such as SEM, AFM,
XRD, and Raman spectroscopy. We found that the SPI/rGO nanocom-
posites showed good biocompatibility when graphene content changed
from 2.5 to 500 μg/mL. The photothermal tests indicated that the
temperature of the SPI/rGO nanocomposite solutions was able to
increase as high as 80 °C after 5 min of irradiation with 808 nm NIR
laser. The more SPI added when forming the nanocomposite, the higher
the temperature it can be reached. Finally, the PTT tests on HeLa cells
indicated that these SPI/rGO nanocomposites exhibited much better
photothermal effects than that of pure GO. We believe that the biocom-
patible SPI/rGO nanocomposite we generated for this study may have
great potential as a photothermal agent for cancer treatment.
Acknowledgments
This work is supported by the National Natural Science Foundation
of China (No. 21274028, 21574023 and 21574024). The authors
sincerely thank Dr. Yuhong Yang, Dr. Shengjie Ling, Dr. Suhang Wang,
Mr. Yingxin Liu, and Mr. Cheng Zhang for their valuable suggestions
and discussions.
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